Use of microRNAs to control virus helper nucleic acids

ABSTRACT

Provided herein are helper nucleic acids comprising at least one microRNA target sequence of an endogenous, cellular microRNA and a nucleic acid encoding a viral protein, wherein the microRNA target sequence is located in the untranslated or translated region of the nucleic acid encoding the viral protein. Also provided are vector systems, compositions and cells comprising the provided helper nucleic acids and a vector or replicon. Methods of making virus-like replicon particles and populations of virus-like replicon particles (VRP) are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/118,954, filed on Dec. 1, 2008, which is incorporated by reference inits entirety.

BACKGROUND

Modern strategies for vaccination and gene therapy often involve the useof viral vectors. This is based on the fact that viruses have evolveduseful techniques for invading the host and self-propagating. The goalis to harness those techniques to deliver immunizing antigens from atarget disease organism or virus, while crippling the virus itself sothat it cannot propagate and sicken its host. The simplest strategy hasbeen the use of live, attenuated viruses, but this solution is obviouslylimited to vaccines for viral diseases. Even so, there is a concern thatsuch attenuated viruses may mutate and become more virulent in the host.A more directed strategy is to design a vector based on a virus, butprovide only the elements necessary for the virus to replicate within acell, rather than propagate and spread throughout the host. Alphaviruseshave been an attractive type of virus to use to design such a system.Flaviviruses, herpesviruses, lentiviruses and adenoviruses are othersuch systems.

All of these single- or restricted-cycle viruses utilize “helper”systems which separately provide some function of the parental virusfrom which they are derived. These helper systems are necessary toproduce the single- or restricted-cycle virus particles, but ideallythey are not carried along with those particles or recombined with thenucleic acids in the particles. However, whenever all portions of aviral genome are present in a cell at the same time, even if onlytransiently, there is the possibility that these elements will recombineto produce the parental virus.

SUMMARY OF THE DISCLOSURE

Provided herein are helper nucleic acids comprising at least onemicroRNA target sequence of an endogenous, cellular microRNA and anucleic acid encoding a viral protein, wherein the microRNA targetsequence is located in the untranslated or translated region of thenucleic acid encoding the viral protein. The viral protein is astructural protein or a protein essential for replication of the virus.Optionally, the viral protein is an alphavirus structural protein. Alsoprovided are vector systems, compositions and cells comprising theprovided helper nucleic acids and a vector or replicon.

Provided are methods of making virus-like replicon particles (VRP)(e.g., alphavirus-like replicon particles (ARP)) comprising transfectinga cell with a replicon, wherein the replicon comprises a packagingsignal, and one or more helper nucleic acids described herein. Theproteins necessary to make the VRP are encoded by one or more of thecell, the replicon or the helper nucleic acid. The cell is then culturedunder conditions that allow for production of assembled virus-likereplicon particles comprising the replicon.

Populations of alphavirus-like replicon particles (ARP) comprising (i) afirst subset of particles comprising a replicon and (ii) a second subsetof particles comprising the one or more provided helper nucleic acids,or a fragment thereof, and a replicon, are provided.

The details of one or more of the compositions and methods are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the location of miRNA targetsengineered into the 3′ UTR of a nucleic acid encoding a structuralprotein. A nucleotide length of 1533 base pairs can occur when thestructural protein is, for example, the capsid protein. The nucleotidelength of 1533 base pairs is measured from the end of the T7 promoter tothe NotI restriction site.

FIG. 2 is a schematic representation of the location of miRNA targetsengineered into the 5′ UTR of a nucleic acid encoding a structuralprotein. A nucleotide length of 1533 base pairs can occur when thestructural protein is, for example, the capsid protein. The nucleotidelength of 1533 base pairs is measured from the end of the T7 promoter tothe NotI restriction site.

FIG. 3 is a schematic representation of the location of miRNA targetsengineered into the 5′ and 3′ UTR of a nucleic acid encoding astructural protein. A nucleotide length of 1647 base pairs can occurwhen the structural protein is, for example, the capsid protein. Thenucleotide length of 1647 base pairs is measured from the end of the T7promoter to the NotI restriction site.

FIG. 4 is a Western blot of capsid and glycoprotein (gp) proteinexpression using capsid and gp helpers with matched microRNA targetsequences. Vero cells were electroporated with sets of three RNAs asfollows: (i) a replicon vector, with capsid helper comprising miRNAtargets RC1-6 and gp helper comprising miRNA targets RC1-6, or (ii) areplicon vector, with capsid and gp helpers comprising either the miRNARC1-3 targets or the miRNA RC4-6 targets. Each of the helpers andreplicon combinations were electroporated in the presence and absence ofmiRNA inhibitors (2′-O-methylated RNA oligonucleotides) specific for thecomplete complement of miRNA targets present on the helpers. The helperRNA combinations with miRNA targets RC1-3 or RC4-6 were alsoelectroporated in the presence of the non-matched miRNA inhibitors todemonstrate the specificity of the inhibitors used. The electroporatedcells were seeded into media and incubated overnight. After incubation(˜18 hours), Venezuelan equine encephalitis virus replicon particles(VEE RPs) were collected.

FIG. 5 is a Northern blot of capsid and gp RNA replication using capsidand gp helpers with matched microRNA target sequences and inhibitors.Samples were prepared as described for FIG. 4.

FIG. 6 is a graph showing VEE RP yields using capsid and gp helpers withmatched microRNA target sequences and inhibitors. Samples were preparedas described for FIG. 4.

FIG. 7 is a Western blot of capsid and glycoprotein (gp) proteinexpression using capsid and gp helpers with non-matched microRNA targetsequences and inhibitors. Combinations of a capsid helper with miRNARC1-3 target sequences and a gp helper with miRNA RC4-6 target sequences(and vice-versa) were electroporated in the presence and absence of all6 miRNA inhibitors (2′-O-methylated RNA oligonucleotides) specific forthe complete complement of miRNA targets present on both helpers. Theelectroporated cells were seeded into media and incubated overnight.After incubation (˜18 hours), VEE RPs were collected.

FIG. 8 is a Northern blot of capsid and gp RNA replication using capsidand gp helpers with non-matched microRNA target sequences andinhibitors. Samples were prepared as described for FIG. 7.

FIG. 9 is a graph showing VEE RP yields using capsid and gp helpers withnon-matched microRNA target sequences and inhibitors. Samples wereprepared as described for FIG. 7.

FIG. 10 is a Western blot of capsid protein expression using capsidhelpers comprising miRNA targets RC1-6 in the 5′ UTR of the capsidhelper RNA on both the plus-strand and minus-strand RNA templates forreplication and in the 3′ UTR on the minus-strand RNA template.Combinations of replicon RNA, unmodified GP helper RNA and one of themiRNA RC1-6 targeted capsid helper RNAs were electroporated into Verocells in the presence and absence of miRNA inhibitors specific for themiRNA targets present on the particular capsid and gp helpers. Theelectroporated cells were seeded into media and incubated overnight.After incubation (˜18 hours), VEE RPs were collected. The miRNA targetlocation on the capsid helper (5′ or 3′ UTR) and the polarity of the RNA(+ or −) are indicated for each lane.

FIG. 11 is a Northern blot of capsid RNA replication using capsidhelpers with miRNA targets RC1-6 in the 5′ UTR of the capsid helper RNAon both the plus-strand and minus-strand RNA templates for replication,and in the 3′ UTR on the minus-strand RNA template. Samples wereprepared as described for FIG. 10.

FIG. 12 is a graph showing VEE RP yields produced using the differentmiRNA targeted capsid helpers as described for FIG. 10.

FIG. 13 is a Western blot of capsid protein expression using capsidhelpers containing six copies of each individual target miRNA sequencecloned into the 3′ UTR of the capsid helper plus-strand RNA. Capsidhelper RNA and unmodified GP helper RNA were combined with replicon RNAand electroporated into Vero cells in the presence or absence of miRNAinhibitors specific for the miRNA targets present on the capsid helper.The electroporated cells were seeded into media and incubated overnight.After incubation (˜18 hours), VEE RPs were collected.

FIG. 14 is a Northern blot of capsid RNA replication using capsidhelpers containing six copies of each individual target miRNA sequencecloned into the 3′ UTR of the capsid helper plus-strand RNA. Sampleswere collected as described for FIG. 13.

FIG. 15 is a graph showing VEE RP yields produced using the differentmiRNA targeted capsid helpers as described for FIG. 13.

FIG. 16 is a Western blot of capsid and gp protein expression usingcapsid and gp helpers constructed with either a single copy, threecopies or six copies of let-7 miRNA target sequence (also referred toherein as miRNA targets) engineered into the 3′ UTR of the plus-strandRNA. Capsid and gp helper RNAs, with the same number of copies of miRNAtarget sequences, were combined with replicon RNA and electroporatedinto Vero cells in the presence or absence of miRNA inhibitors specificfor the miRNA targets present on the helpers. In one set ofelectroporations, the 2′-β-methylated oligonucleotide miRNA inhibitorspecific for let-7 was replaced with a phosphorodiamidate morpholinooligomer (PMO) specific for let-7 miRNA. The electroporated cells wereseeded into media and incubated overnight. After incubation (˜18 hours),VEE RPs were collected.

FIG. 17 is a Northern blot of capsid and gp RNA replication using capsidand GP helpers constructed with either a single copy, three copies orsix copies of the let-7 miRNA target sequence engineered into the 3′ UTRof the plus-strand RNA. Samples were collected as described for FIG. 16.

FIG. 18 is a graph showing VEE RP yields produced using the differentmiRNA targeted helpers as described for FIG. 16.

FIG. 19 is a Western blot of capsid and gp protein expression usingcapsid and GP helpers constructed with either a single copy, threecopies or six copies of miR-17 miRNA target sequence (also referred toas RC 5) engineered into the 3′ UTR of the plus-strand RNA. Capsid andgp helper RNAs, with the same number of copies of this miRNA targetsequence, were combined with replicon RNA and electroporated into Verocells in the presence or absence of miRNA inhibitors specific for themiRNA targets present on the helpers. The electroporated cells wereseeded into media and incubated overnight. After incubation (˜18 hours),VEE RPs were collected.

FIG. 20 is a Northern blot of capsid and gp RNA replication using capsidand GP helpers constructed with either a single copy, three copies, orsix copies of the mir-155 miRNA target sequence engineered into the 3′UTR of the plus-strand RNA. Samples were collected as described for FIG.19.

FIG. 21 is a graph showing VEE RP yields produced using the differentmiRNA targeted helpers as described for FIG. 19.

FIG. 22 is a graph of the results of HA-specific ELISPOT analysis of VRPpreparations produced and tested in BALB/c mice. Combinations of bothmiRNA targeted (“miCap”, “miGP”, “miCap+miGP”) helpers were mixed withunmodified (“WT”) or miRNA target-modified replicon vectors (“miREP”)expressing the influenza HA gene as shown in Table 3. Groups of 16 micewere immunized with equivalent doses of each of the VEE RP. Seven daysafter the priming dose, half of the mice (8) were sacrificed, thesplenocytes were collected and HA-specific gamma interferon ELISPOTanalysis was conducted.

FIG. 23 is a graph of the results of HA-specific ELISPOT analysis of VRPpreparations as prepared for FIG. 22 administered to BALB/c mice threeweeks after the priming dose, the remaining mice in each group wereboosted with their respective HA VRP. Seven days after the boost theremaining animals were sacrificed and the splenocytes were collected forHA-specific gamma interferon ELISPOT analysis.

FIG. 24 is a graph of HA-specific ELISA analysis of serum samplesobtained from the BALB/c mice immunized as described for FIG. 22.

FIG. 25 is a graph of HA-specific ELISA analysis of serum samplesobtained from the BALB/c mice immunized as described for FIG. 23.

FIG. 26 is a graph of the VEE RP neutralization titers determined frommice vaccinated with VEE RP packaged with different helper combinations(see Table 6 for details). Serum samples were collected from mice sevendays after the boosting vaccination. The titer for each group of animalsrepresents the mean of the last dilution at which the serum was able toneutralize 80% of a preparation of green fluorescent protein expressingVEE RP.

DETAILED DESCRIPTION

Provided herein are methods of exploiting microRNA, also referred to asmiRNA, regulatory function to control aspects of replication ofviral-based vector systems (such as alphavirus replicon-helper systems).Although the present disclosure focuses on the example of alphaviruses,it is contemplated that other viral-based vector systems including, forexample, lentiviral, herpesviral and adenoviral vector systems, can bemodified as described herein for alphaviruses.

The Alphavirus genus includes a variety of viruses, all of which aremembers of the Togaviridae family. The studies of these viruses have ledto the development of techniques for vaccinating against the alphavirusdiseases and against other diseases through the use of alphavirusvectors for the introduction of foreign genes and also for gene therapyapplications. One strategy involving alphavirus vectors is the insertionof sequences encoding immunizing antigens of pathogenic agents into alive, replicating strain of an alphavirus vector. Another strategy is toutilize a replicon vector to express the immunizing antigens. Suchalphavirus replicon vectors can be packaged into alphavirus-likereplicon particles, referred to herein as ARPs, by supplying thestructural protein genes in trans. The structural proteins can beprovided in a number of ways. The structural proteins can be provided bypackaging cell lines engineered to express the proteins either in aconstitutive or inducible manner (Polo et al., Proc. Natl. Acad. Sci.USA 96(8):4598-603 (1999)). The structural proteins can be expressedtransiently from DNA plasmids under the control of a polymerase IIpromoter or they can be provided on helper RNAs that are replicated bythe replicon vector itself (Pushko et al., Virology 239(2):389-401(1997); Rayner et al., Rev. Med. Virol. 12:279-296 (2002)). Each ofthese approaches ultimately results in production of structural proteinmRNAs, and translation of these mRNAs, in the cytoplasm of the cell usedto package the replicon RNA. It has been demonstrated that replicationcompetent alphaviruses can be generated by co-packaging of helper RNA orrecombination of helper RNAs with replicon RNA (Geigenmuller-Gnirke etal., Proc. Natl. Acad. Sci. USA 88(8):3253-7 (1991); Weiss andSchlesinger, J. Virol. 65(8):4017-25 (1991); Raju et al., J. Virol.69(12):7391-401 (1995); and Hill et al., J. Virol. 71(4):2693-704(1997)). A significant advance in reducing the probability of generatingreplication competent alphaviruses was described when the structuralprotein genes were separated onto two different RNA helpers. Twoseparate recombination events are required to reconstitute a completealphavirus genome when using a two helper RNA system. The probability ofthis event occurring is significantly lower than when using a singlehelper RNA system (Pushko et al., Virology 239(2):389-401 (1997); Frolovet al., J. Virol. 71(4):2819-29 (1997); and Smerdou and Liljestrom, J.Virol. 73(2):1092-8 (1999)). However, there are still theoreticalpredictions for replication competent alphaviruses to arise using a twohelper RNA system, for example, there is the possibility that helperRNAs may be co-packaged, even in the absence of a defined packagingsignal in the helper RNA.

MicroRNAs (miRNA) are small RNAs approximately 21-24 nucleotides (nt) inlength that have been identified in cells and are associated withregulation of mRNA translation and stability. Numerous miRNAs have beenidentified that control cellular activities such as proliferation, celldeath, fat metabolism, neuronal patterning in nematodes, hematopoieticdifferentiation and plant development. These small regulatory RNAs havebeen identified in a wide range of animals including mammals, fish,worms and flies. The miRNA sequences are transcribed from specific miRNAgenes, as independent transcriptional units, throughout the genome orare produced in coordination with intron processing of specific mRNAsthus combining control of protein expression with mRNA processing. Thecellular production of miRNAs begins with transcription of largeprecursor primary miRNAs which are processed by a nuclear ribonucleaseIII-like enzyme, Drosha. The large precursor RNAs are termed pri-miRNAsand the smaller Drosha-processed species, termed pre-miRNAs, areexported from the nucleus by Exportin 5. The pre-miRNA, exported fromthe nucleus, is then processed in the cytoplasm by another ribonucleaseIII-like enzyme called Dicer into a mature miRNA. The mature miRNA isthen transferred to the RNA-induced silencing complex (RISC) whichguides the miRNA to its target RNA. The 5′-most 7 to 8 nucleotides(specifically nt 2-8) of the miRNA (sometimes referred to as the seedsequence) are involved in Watson-Crick base pairing with nucleotides inthe 3′ untranslated region (UTR) of the target mRNA. If the base pairingis perfect the target mRNA is cleaved by the RISC endonuclease activity.Alternatively, if the base pairing is imperfect the target mRNA becomestranslationally inactive and protein expression is affected without mRNAdegradation.

Provided herein are alphavirus helper systems that use miRNA elements totarget the helper RNAs in environments that they are not intended to befunctional in (such as in a vaccinated subject). The system designrequires that the action of the endogenous, cellular miRNA on its targetsequence (present on the helper RNA) must be absent or inhibited in thecells used to generate VRP, but be active (i.e., control helperreplication) in cells of the subject. It is possible to inhibit cellularmiRNAs by treating cells in which the helper function is desired (e.g.,packaging cell lines such as Vero) with complementary RNA sequencesspecific for the miRNA of interest. There are at least 4 types ofinhibitory RNAs that can be used to inhibit cellular miRNAs, and one ormore of these can be introduced to the cell as provided in the methodsdescribed herein Inhibitory RNAs include, for example, modifiedoligonucleotides (2′-O-methylated or 2′-O-methoxyethyl), locked nucleicacids (LNA; see, e.g, Válóczi et al., Nucleic Acids Res. 32(22):e175(2004)), morpholino oligonucleotides (see, e.g, Kloosterman et al., PLoSBiol 5(8):e203 (2007)), peptide nucleic acids (PNAs), PNA-peptideconjugates, LNA/2′-O-methylated oligonucleotide mixmers (see, e.g.,Fabiani and Gait, RNA 14:336-46 (2008)) and mRNAs with multiple copiesof the miRNA target engineered into the 3′ UTR, sometimes referred to asmiRNA sponges. The miRNA inhibitors function by sequestering thecellular miRNAs away from the mRNAs that normally would be targeted bythem. As described herein, the miRNA inhibitors are added to the cellsin which VRP will be produced from miRNA-targeted helpers and repliconRNA. The miRNA inhibitors suppress the miRNA control of targeted helperreplication, thereby allowing normal viral structural protein expressionfrom the helper(s) and packaging of the replicon RNA into VRP. The useof miRNA-targeted helper RNAs provides control of not only co-packagedhelper RNAs but also of helper RNAs that have recombined with thereplicon RNA. The latter would be accomplished because, as long as themiRNA target is maintained after the replicon-helper recombinationevent, this recombinant RNA molecule would still be targeted for controlvia the RNAi pathway described above.

Provided herein are methods of exploiting microRNA regulatory functionto control aspects of replication of virus-based vector or repliconsystems, for example, alphavirus-based vector or replicon systems. TheAlphavirus genus includes a variety of viruses, all of which are membersof the Togaviridae family. As used herein, the term alphavirus includesvarious species such as, for example Eastern Equine Encephalitis Virus(EEE), Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus,Mucambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEE),Sindbis Virus, Semliki Forest Virus, Middleburg Virus, ChikungunyaVirus, O'nyong-nyong Virus, Ross River Virus, Barmah Forest Virus, GetahVirus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, AuraVirus, Whataroa Virus, Babanki Virus, Kyzylagach Virus, Highlands Jvirus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus. Thealphaviral genome is a single-stranded, messenger-sense RNA, modified atthe 5′-end with a methylated cap and at the 3′-end with avariable-length poly (A) tract. Structural subunits containing a singleviral protein, capsid, associate with the RNA genome in an icosahedralnucleocapsid. In the virion, the capsid is surrounded by a lipidenvelope covered with a regular array of transmembrane protein spikes,each of which consists of a heterodimeric complex of two glycoproteins,E1 and E2. See, e.g., Pedersen et al., J. Virol. 14:40 (1974). Sindbisvirus and Semliki Forest virus (SFV) are considered the prototypicalalphaviruses and have been studied extensively. See Schlesinger, TheTogaviridae and Flaviviridae, Plenum Publishing Corp., New York (1986).The VEE virus has been studied extensively, see, e.g., U.S. Pat. No.5,185,440 and references cited therein. Alphaviruses useful in theconstructs and methods provided herein include, but are not limited to,VEE, S.A. AR86, Sindbis (e.g., TR339, see U.S. Pat. No. 6,008,035), andSFV.

Alphavirus vector or replicon systems have been described. See, e.g.,U.S. Pat. No. 5,185,440 to Davis et al., International Publication No.WO 92/10578; U.S. Pat. Nos. 5,505,947 and 5,643,576 to Johnston et al.;Hahn et al., Proc. Natl. Acad. Sci. USA 89:2679-83 (1992); U.S. Pat. No.6,190,666 to Garoff et al.; U.S. Pat. Nos. 5,792,462; 6,156,558;6,521,235; 6,531,135; 6,541,010 and 7,235,235 to Johnston et al.; U.S.Pat. Nos. 7,045,335 and 7,078,218 to Smith et al.; U.S. Pat. Nos.5,814,482, 5,843,723, 5,789,245, 6,015,694, 6,105,686 and 6,376,236 toDubensky et al.; U.S. Patent Publication No. 2002-0015945 by Polo etal.; U.S. Patent Publication No. 2001-0016199 by Johnston et al.; U.S.Patent Publication No. 2005-0266550 by Rayner et al.; Frolov et al.,Proc. Natl. Acad. Sci. USA 93:11371-7 (1996); Pushko et al., Virology239:389-401 (1997); Polo et al., Proc. Natl. Acad. Sci. USA96(8):4598-603 (1999); Rayner et al., Rev. Med. Virol. 12:279-96 (2002);Geigenmuller-Gnirke et al., Proc. Natl. Acad. Sci. USA 88(8):3253-7(1991); Weiss and Schlesinger, J. Virol. 65(8):4017-25 (1991); Raju etal., J. Virol. 69(12):7391-401 (1995); Hill et al., J. Virol.71(4):2693-704 (1997); Frolov et al., J. Virol. 71(4):2819-29 (1997);and Smerdou and Liljestrom, J. Virol. 73(2):1092-8 (1999). Such repliconsystems include one or more helper nucleic acid and one or morereplicon. As an example, endogenous, cellular miRNA target sequences areincorporated into 1) capsid helper RNAs, 2) glycoprotein helper RNAs 3)or replicon vector RNAs. Naturally occurring endogenous, cellular miRNAssignificantly inhibit the ability of the target-sequence containinghelper nucleic acids to replicate in vitro and in vivo. The use ofendogenous, cellular miRNA target sequences that are tissue-specificand/or development stage-specific in the replicon vector RNAs providescontrol of replicon expression only in tissues or at stages which aredesired. As used herein, the phrase endogenous, cellular microRNA ormiRNA refers to a microRNA encoded by a cell of an organism other than avirus. The organism can be unicellular or multi-cellular. Thus, themicroRNA can be encoded by a cell of a prokaryote or eukaryote. Thus,the microRNA can be encoded by a cell of an animal, such as for example,a mammal or human.

Provided herein are helper nucleic acids comprising a 5′ alphavirusrecognition sequence; a nucleic acid sequence encoding an alphavirusstructural protein; a 3′ alphavirus replication recognition sequence;and at least one microRNA target sequence of an endogenous, cellularmicroRNA. Optionally, the helper nucleic acids can comprise at least onemicroRNA target sequence of an endogenous, cellular microRNA and anucleic acid sequence encoding an alphavirus structural protein, whereinthe microRNA target sequence is located in a region of the nucleic acidencoding the alphavirus structural protein. A region of the nucleic acidencoding the alphavirus structural protein can, for example, include thetranslated or untranslated region (UTR) (e.g., a 3′- or 5′-UTR) of thenucleic acid. The terms alphavirus helper(s), alphavirus helper nucleicacid(s), or alphavirus helper construct(s), refer to a nucleic acidmolecule that is capable of expressing one or more alphavirus structuralproteins. Smith et al. (International Patent Publication WO2004/085660), Smith et al. (U.S. Pat. No. 7,045,335), and Kamrud et al.(U.S. Patent Publication No. 2009-0075384) describe numerous helperconstructs useful for expressing alphavirus structural proteins in theproduction of ARPs.

The terms 5′ alphavirus replication recognition sequence and 3′alphavirus replication recognition refer to the RNA sequences found inalphaviruses, sequences derived therefrom, or synthetic sequences basedon conserved sequence among various alphaviruses, that are recognized bythe nonstructural alphavirus replicase proteins and lead to replicationof viral RNA. These sequences can, for example, be in the form of DNA tofacilitate the preparation, mutation and/or manipulation of the helpernucleic acids described herein. The use of 5′ and 3′ replicationrecognition sequences results in the replication and/or transcription ofthe RNA sequence encoded between the two sequences.

The microRNA target sequence of the helper nucleic acid is located, forexample, in the translated region, the 5′ UTR, or the 3′ UTR of thenucleic acid encoding the alphavirus structural protein. Optionally,multiple microRNA targets are located in different locations of thenucleic acid; e.g. at least one target sequence is located in the 3′ UTRof the nucleic acid encoding the alphavirus structural protein and atleast one target sequence is located in the 5′ UTR of the nucleic acidencoding the alphavirus structural protein. When located in thetranslated region of the helper nucleic acid, the microRNA targets areoptionally designed to be in-frame and to encode amino acids. However,the additional amino acids do not significantly affect the level ofprotein expression or its function. For example, the microRNA targetscan be inserted in the leader sequence of the structural protein. In thecase of alphavirus, the microRNA targets can be inserted into thesequence, referred to as “E3” (see below). It has been shown previouslythat additional amino acids may be inserted in E3 without detrimentaleffect (see, e.g., London et al., Proc. Natl. Acad. Sci. USA 89:207-11(1992)). Optionally, the sequence of the translated region of thealphavirus structural protein can be altered to create a microRNA targetsequence without changing the amino acid number or content of theprotein. For example, conservative substitutions could be made in thenucleotide sequence of the alphavirus structural protein using theredundancy of the codon-assignments without altering the amino acidencoded by the codon. Optionally, the helper nucleic acid comprises twoor more microRNA target sequences. Thus, the helper nucleic acid cancomprise two, three, four, five, six, seven, eight, nine, ten, or moremicroRNA target sequences. Optionally, the helper nucleic acid comprises1 to 10, 1 to 20, 1 to 30, 5 to 10, 5 to 20, 5 to 30, 10 to 20, 10 to30, or 15 to 30 microRNA target sequences. Optionally, the microRNAtarget sequence has 100% complementarity to the endogenous, cellularmicroRNA. Alternatively, the microRNA target sequence has less than 100%complementarity to the endogenous, cellular microRNA. A percentcomplementarity indicates the percentage of residues in a nucleic acidmolecule that can form hydrogen bonds (e.g., Watson-Crick base pairing)with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10nucleotides out of a total of 10 nucleotides in the firstoligonucleotide being based paired to a second nucleic acid sequencehaving 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100%complementary respectively). 100% complementary means that all theresidues of a nucleic acid sequence will hydrogen bond with the samenumber of residues in a second nucleic acid sequence. Thus, the microRNAtarget sequence has 100%, 95%, 90%, 85%, 80%, 75%, 70% complementarity,or any percent complementarity between 100% and 70%, to the endogenous,cellular microRNA. Optionally, a portion of the microRNA targetsequence, for example, the 5′-8 nucleotides, which is often referred toas the “seed” sequence, is identical (i.e., has 100% complementary) tothe endogenous, cellular microRNA, while a second portion of themicroRNA target sequence has less than 100% complementarity, e.g. 50%,to the endogenous, cellular microRNA. Optionally, the size of themicroRNA target sequence is 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50,10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 30, 15 to 40, 15to 50, 20 to 25, 20 to 30, 20 to 40, 20 to 50, 25 to 30, 25 to 40, or 25to 50 nucleotides.

As discussed above, the helper nucleic acid comprises one or morealphavirus structural proteins. Thus, the helper nucleic acid comprisesone or more than one alphavirus structural protein. As used herein, theterms alphavirus structural protein/protein(s) refers to one or acombination of the structural proteins encoded by alphaviruses. Forexample, the alphavirus structural protein is a Venezuelan equineencephalitis (VEE) virus structural protein. By way of another example,the alphavirus structural protein is selected from the group consistingof South African Arbovirus No. 86, Sindbis virus, Semliki Forest Virus,and Ross River Virus structural proteins. Alphavirus structural proteinsare produced by the alphavirus as a polyprotein and are representedgenerally in the literature as C-E3-E2-6k-E1. E3 and 6k serve asmembrane translocation/transport signals for the two glycoproteins, E2and E1. Thus, use of the term E1 herein can refer to E1, E3-E1, 6k-E1,or E3-6k-E1, and use of the term E2 herein can refer to E2, E3-E2,6k-E2, or E3-6k-E2. Typically, alphavirus structural proteins includethe capsid protein, E1 glycoprotein, and E2 glycoprotein in the maturealphavirus (certain alphaviruses, such as Semliki Forest Virus, containan additional protein, E3, in the mature coat). Thus, as an example, thehelper nucleic acid comprises an alphavirus capsid protein. Optionally,the alphavirus capsid protein is a VEE capsid protein. As anotherexample, the helper nucleic acid comprises an alphavirus glycoprotein.Optionally, the alphavirus glycoprotein is a VEE glycoprotein. Aglycoprotein, as referred to herein, can be encoded by a nucleic acidsequence comprising a single open reading frame (orf) encodingE3-E2-6k-E1, an orf encoding E3-E2, an orf encoding 6k-E1, an orfencoding E3-E1, an orf encoding E3-6k-E1, an orf encoding 6k-E2, or anorf encoding E3-6k-E2.

The structural proteins of the alphavirus are distributed among one ormore helper nucleic acid molecules (e.g., a first helper RNA (or DNA)and a second helper RNA (or DNA)). The same structural protein can beencoded by more than one helper nucleic acid. The helper nucleic acidsencode at least one but not all alphavirus structural proteins, and asecond helper nucleic acid encodes at least one alphavirus structuralprotein not encoded by a first helper nucleic acid. In addition, one ormore structural proteins may be located on the same molecule as thereplicon nucleic acid, provided that at least one alphavirus structuralprotein is not located on the replicon RNA such that the replicon andresulting alphavirus particle are propagation defective. As used herein,the term not located on means either total lack of the specified segmentor the lack of a sufficient portion of the specified segment to renderthe segment inoperative or nonfunctional, in accordance with standardusage. See, e.g., U.S. Pat. No. 4,650,764 to Temin et al. The termreplication defective as used herein is synonymous with propagationdefective, and means that the particles contacted with a given host cellcannot produce progeny particles in the host cell, due to the absence ofthe helper function, i.e. the alphavirus structural proteins requiredfor packaging the replicon nucleic acid. However, the replicon nucleicacid is capable of replicating itself and being expressed within thehost cell into which it has been introduced.

In all of the helper nucleic acids described herein, it is understoodthat these molecules further comprise sequences necessary for expression(encompassing translation and where appropriate, transcription orreplication signals) of the encoded structural protein sequences in thehelper cells. Such sequences may include, for example, promoters,(either viral, prokaryotic or eukaryotic, inducible or constitutive),IRES elements, and 5′ and 3′ viral replicase recognition sequences.Optionally, one or more helper nucleic acids may include only the 5′ and3′ viral replication recognition sequences (see Kamrud et al., U.S.Patent Publication No. 2009-0075384), thus functioning directly asreplicable mRNAs when introduced into a eukaryotic cell. In the case ofthe helper nucleic acids expressing one or more glycoproteins, it isunderstood that these sequences are advantageously expressed with aleader or signal sequence at the N-terminus of the structural proteincoding region in the nucleic acid constructs. The leader or signalsequence is derived from the alphavirus, for example E3 or 6k, or it isa heterologous sequence such as a tissue plasminogen activator signalpeptide or a synthetic sequence. Thus, as an example, a first helpernucleic acid may be an RNA molecule encoding capsid-E3-E1, and thesecond helper nucleic acid may be an RNA molecule encoding capsid-E3-E2.Alternatively, the first helper RNA can encode capsid alone, and thesecond helper RNA can encode E3-E2-6k-E1. Additionally, packagingsignal(s) or encapsidation sequence(s) that may be present in the viralgenome are optionally not present in all of the helper nucleic acids.Thus, packaging signal(s) are, optionally, not located on all of thehelper nucleic acids.

The helper nucleic acids described herein can be introduced intopackaging cells, i.e. those cells in which a replicon is packaged by thestructural proteins expressed from the helpers, in a number of ways.They can be expressed from one or more expression cassettes that havebeen stably transformed into the cells, thereby establishing packagingcell lines (see, e.g., U.S. Pat. No. 6,242,259). Alternatively, thehelper nucleic acid is incorporated into the packaging cell genome priorto the introduction/expression of the RNA replicon vector. Optionally,the helper nucleic acid contains an inducible promoter such thatexpression of the structural proteins is induced with the appropriatestimulus just prior to, concomitant with, or after the introduction ofthe RNA replicon vector.

Alternatively, the RNAs can be introduced as RNA or DNA molecules thatcan be expressed in the helper cell without integrating into the cellgenome. Methods of introduction include electroporation, viral vectors(e.g., SV40, adenovirus, nodavirus, astrovirus), and lipid-mediatedtransfection.

An alternative to multiple helper RNAs is the use of a single DNAmolecule, which encodes all the polypeptides necessary for packaging theviral replicon RNA into infective alphavirus replicon particles (see,e.g., U.S. Pat. No. 7,045,335). Thus, as one example, the helper nucleicacid is an RNA. As another example, the helper nucleic acid is a DNA.The single DNA helper is introduced into the packaging cell by any meansknown including, but not limited to, electroporation, lipid-mediatedtransfection (lipofection), viral vector (e.g., adenovirus or SV-40), orcalcium phosphate-mediated transfection. The DNA is typicallyelectroporated into cells with a decreased voltage and increase incapacitance, as compared to that required for uptake of RNA. Thus, theconditions for electroporation of DNA into cells may vary from thoserequired for the uptake of RNA, but can be determined by routineexperimentation. In all electroporations, the value for the voltage andcapacitance must be set so as to avoid destroying the ability of thepackaging (host) cells to produce infective virus particles.

As provided herein and discussed in more detail below, the helpernucleic acids are used in combination with a replicon to producealphavirus or alphavirus-like replicon particles. The termsalphavirus-like replicon particles (ARPs), or recombinant alphavirusparticles, used interchangeably herein, mean a virion-like structuralcomplex incorporating an alphavirus replicon RNA. The repliconoptionally expresses one or more heterologous RNA sequences. Optionally,the virion-like structural complex includes one or more alphavirusstructural proteins embedded in a lipid envelope enclosing anucleocapsid that in turn encloses the RNA. The lipid envelope istypically derived from the plasma membrane of the cell in which theparticles are produced. Optionally, the alphavirus replicon RNA issurrounded by a nucleocapsid structure comprised of the alphaviruscapsid protein, and the alphavirus glycoproteins are embedded in thecell-derived lipid envelope. The structural proteins and replicon RNAmay be derived from the same or different alphaviruses. For example, thereplicon RNA and structural proteins are from VEE, see, e.g., U.S.Patent Publication 2005-0266550 by Smith et al. Optionally, the repliconRNA is derived from VEE and the structural proteins are derived fromSindbis Virus (see, e.g., U.S. Pat. No. 6,376,236 to Dubensky et al.).The alphavirus replicon particles are infectious but propagationdefective, i.e., the replicon RNA cannot propagate beyond the host cellinto which the particles initially infect, in the absence of the helpernucleic acid(s) encoding the alphavirus structural proteins.

Thus, provided is a population of alphavirus-like replicon particles(ARP) comprising (i) a first subset of particles comprising a repliconand (ii) a second subset of particles comprising one or more helpernucleic acids as described herein which comprise one or more miRNAtarget sequences, or a fragment thereof, and a replicon. Optionally, theARPs are derived from Venezuelan equine encephalitis (VEE) virus, SouthAfrican Arbovirus No. 86, Sindbis virus, Semliki Forest Virus, or RossRiver Virus. Optionally, as discussed below, the replicon encodes apolypeptide, an immunostimulatory polypeptide, an immunogenicpolypeptide, or a therapeutic product. A population of alphavirusreplicon particles provided herein contains no detectablereplication-competent virus particles, as determined by passage onpermissive cells in culture. Optionally, the population of alphavirusreplicon particles contains one or more attenuating mutations in eitheran alphavirus structural protein or an alphavirus nonstructural proteinor both an alphavirus structural protein and an alphavirus nonstructuralprotein.

The terms alphavirus RNA replicon, replicon, replicon RNA, alphavirusreplicon RNA, alphavirus RNA vector replicon, are used interchangeablyto refer to an RNA molecule expressing nonstructural polypeptides suchthat it can direct its own replication (amplification) and comprises, ata minimum, 5′ and 3′ viral replication recognition sequences (e.g., 5′and 3′ alphavirus replication recognition sequences), coding sequencesfor viral nonstructural proteins (e.g., alphavirus nonstructuralproteins), and a polyadenylation tract. As used herein, the terms 5′alphavirus replication recognition sequence and 3′ alphavirusreplication recognition sequence refer to the sequences found inalphaviruses, or sequences derived therefrom, that are recognized by thenonstructural alphavirus replicase proteins and lead to replication ofviral RNA. These are sometimes referred to as the 5′ and 3′ ends, oralphavirus 5′ and 3′ sequences. These sequences can be modified bystandard molecular biological techniques, see for example U.S. PatentPublication No. 2007-0166820 and U.S. Patent Publication No.2009-0075384) to further minimize the potential for recombination or tointroduce cloning sites, with the proviso that they must be recognizedby the alphavirus replication machinery. In addition, the repliconoptionally contains one or more elements to direct the expression,meaning transcription and translation, of a heterologous RNA sequence.It is optionally engineered to express alphavirus structural proteins.For example, Smith et al. (International Publication WO 2004/085660) andSmith et al. (U.S. Pat. No. 7,045,335) describe numerous constructs forsuch alphavirus RNA replicons, which are herein incorporated byreference in their entireties.

The alphavirus RNA replicon provided herein is designed to express oneor more heterologous coding sequence(s) or functional RNA(s) ofinterest, also referred to herein as a heterologous RNA or heterologoussequence, which can be chosen from a wide variety of sequences derivedfrom viruses, prokaryotes or eukaryotes, including native, modified orsynthetic antigenic proteins, peptides, epitopes or immunogenicfragments. Thus, the replicon can encode a polypeptide. Suitablepolypeptides include, for example, immunostimulatory molecules.Immunostimulatory polypeptides include, for example, IL-12. Optionally,the replicon encodes an immunogenic polypeptide. Immunogenicpolypeptides include, for example, immunogenic polypeptides derived fromcancer cells, tumor cells, toxins or an infectious agent. Infectiousagents include, for example, viruses, bacteria, fungi and parasites. Asused herein, an immunogenic polypeptide, immunogenic peptide, orimmunogen includes any peptide, protein or polypeptide that elicits animmune response in a subject. Optionally, the immunogenic polypeptide issuitable for providing some degree of protection to a subject against adisease. These terms can be used interchangeably with the term antigen.Optionally, the immunogenic polypeptide can comprise, consistessentially of or consist of one or more epitopes. As used herein, anepitope is a set of amino acid residues which is involved in recognitionby a particular immunoglobulin or immunoglobulin fragment.

Optionally, the replicon encodes a therapeutic product, such as, forexample, a therapeutic protein or an inhibitory nucleic acid. Inhibitorynucleic acid includes an antisense molecule, a triplex formingoligonucleotide, an external guide sequence, an aptamer, an siRNA, anmiRNA, an shRNA and a ribozyme.

Optionally, the replicon does not express a polypeptide or a therapeuticproduct. Such an “empty” replicon can be packaged into a VRP and used asan adjuvant to enhance the immunogenicity of other products, includingother VRPs.

As used herein, expression directed by a particular sequence is thetranscription of an associated downstream sequence. If appropriate anddesired for the associated sequence, then the term expression alsoencompasses translation (protein synthesis) of the transcribed orintroduced RNA. Optionally, transcription and translation of thereplicon, heterologous RNAs, and helper RNAs are controlled separatelyby different regulatory elements. Optionally, control of nucleic acidexpression at the level of translation is accomplished by introducing aninternal ribosome entry site (IRES) downstream of the promoter, e.g.,the alphavirus 26S subgenomic promoter, and upstream of the codingsequence, e.g., for the heterologous sequence or an alphavirusstructural protein, to be translated. This can be referred to as asubgenomic promoter-IRES-heterologous nucleic acid of interest (NOI)cassette. Optionally, a spacer sequence is incorporated in between thealphavirus 26S subgenomic promoter and the IRES element. The spacersequence provides optimal spacing between these two elements to enhancethe translation from the IRES. The IRES element is positioned so that itdirects translation of the mRNA, thereby minimizing, limiting orpreventing initiation of translation of the mRNA from themethyl-7-guanosine (5′)pppN structure present at the 5′ end of thesubgenomic mRNA (the “cap”). Such IRES-directed translation is sometimesreferred to as “cap-independent” translation. These constructs result inthe IRES controlling translation of a heterologous sequenceindependently of promoter-driven transcription (See, e.g., U.S. Pat. No.7,442,381 to Smith et al.). IRES elements from many different sourcescan be employed, including viral IRES elements from picornaviruses,e.g., poliovirus (PV) or the human enterovirus 71, e.g. strains7423/MS/87 and BrCr thereof; from encephalomyocarditis virus (EMCV);from foot-and-mouth disease virus (FMDV); from flaviviruses, e.g.,hepatitis C virus (HCV); from pestiviruses, e.g., classical swine fevervirus (CSFV); from retroviruses, e.g., murine leukemia virus (MLU); fromlentiviruses, e.g., simian immunodeficiency virus (SIV); from cellularmRNA IRES elements such as those from translation initiation factors,e.g., eIF4G or DAP5; from transcription factors, e.g., c-Myc orNF-κB-repressing factor (NRF); from growth factors, e.g., vascularendothelial growth factor (VEGF), fibroblast growth factor (FGF-2) andplatelet-derived growth factor B (PDGF B); from homeotic genes, e.g.,Antennapedia; from survival proteins, e.g., X-linked inhibitor ofapoptosis (XIAP) or Apaf-1; from chaperones, e.g., immunoglobulinheavy-chain binding protein BiP, plant viruses, as well as any otherIRES elements. The term transcription as used herein includes theproduction of RNA from a recombinant replicon or helper nucleic acid,which can itself be an RNA molecule.

Optionally, promoterless helpers can be employed. Such helper moleculesdo not contain a promoter; rather, they are introduced as replicableRNAs comprising 5′ and 3′ replication recognition sequences. Translationof promoterless helper nucleic acids occurs via the 5′ cap on the RNAmolecule. Optionally, in the absence of any promoter element on thehelper nucleic acid, an IRES element may be included to directtranslation.

Optionally, one or more of the nucleic acids encoding the alphavirusstructural proteins, i.e., the capsid, E1 glycoprotein and E2glycoprotein, or the replicon construct, contain one or more attenuatingmutations. An attenuating mutation refers to a nucleotide deletion,addition, or substitution of one or more nucleotide(s), or a mutationthat comprises rearrangement or chimeric construction which results in aloss of virulence in a live virus containing the mutation as compared tothe appropriate wild-type virus. The phrases attenuating mutation andattenuating amino acid, as used herein, also mean a nucleotide mutationthat may or may not be in a region of the viral genome encodingpolypeptides or an amino acid coded for by a nucleotide mutation. In thecontext of a live virus, attenuating mutations result in a decreasedprobability of the alphavirus causing disease in its host (i.e., a lossof virulence), in accordance with standard terminology, whether themutation be a substitution mutation, or an in-frame deletion or additionmutation. See, e.g., Davis et al., Microbiology, 4^(th) Ed., 156-158(1990). The phrase attenuating mutation excludes mutations which wouldbe lethal to the virus, unless such a mutation is used in combinationwith a restoring mutation which renders the virus viable, albeitattenuated. Methods for identifying suitable attenuating mutations inthe alphavirus genome are known. Olmsted et al., describes a method ofidentifying attenuating mutations in Sindbis virus by selecting forrapid growth in cell culture (Olmsted et al., Science 225:424 (1984)).Johnston and Smith, describe the identification of attenuating mutationsin VEE by applying direct selective pressure for accelerated penetrationof BHK cells (Johnston and Smith, Virology 162:437 (1988)). Attenuatingmutations in alphaviruses have been described in the art, e.g. White etal., J. Virology 75:3706 (2001); Kinney et al., Virology 70:19 (1989);Heise et al., J. Virology 74:4207 (2000); Bernard et al., Virology276:93 (2000); Smith et al., J. Virology 75:11196 (2001); Heidner andJohnston, J. Virology 68:8064 (1994); Klimstra et al., J. Virology73:10387 (1999); Glasgow et al., Virology 185:741 (1991); Polo andJohnston, J. Virology 64:4438 (1990); and Smerdou and Liljestrom, J.Virology 73:1092 (1999).

Appropriate attenuating mutations depend upon the alphavirus used. Forexample, when the alphavirus is VEE, suitable attenuating mutationsinclude those selected from the group consisting of codons at E2 aminoacid position 76 which specify an attenuating amino acid, preferablylysine, arginine, or histidine as E2 amino acid 76; codons at E2 aminoacid position 120 which specify an attenuating amino acid, preferablylysine as E2 amino acid 120; codons at E2 amino acid position 209 whichspecify an attenuating amino acid, preferably lysine, arginine, orhistidine as E2 amino acid 209; codons at E1 amino acid 272 whichspecify an attenuating mutation, preferably threonine or serine as E1amino acid 272; codons at E1 amino acid 81 which specify an attenuatingmutation, preferably isoleucine or leucine as E1 amino acid 81; andcodons at E1 amino acid 253 which specify an attenuating mutation,preferably serine or threonine as E1 amino acid 253. Additionalattenuating mutations include deletions or substitution mutations in thecleavage domain between E3 and E2 such that the E3/E2 polyprotein is notcleaved; this mutation in combination with the mutation at E1-253 is apreferred attenuated strain for use in this invention. Similarly,mutations present in existing live vaccine strains, e.g. strain TC83(see, e.g., Kinney et al., Virology 170:19-30 (1989), particularly themutation at nucleotide 3), are also optionally employed in the particlesmade by the provided methods.

Where the alphavirus is the South African Arbovirus No. 86 (S.A. AR86),suitable attenuating mutations include those selected from the groupconsisting of codons at nsP1 amino acid position 538 which specify anattenuating amino acid, preferably isoleucine as nsP1 amino acid 538;codons at E2 amino acid position 304 which specify an attenuating aminoacid, preferably threonine as E2 amino acid position 304; codons at E2amino acid position 314 which specify an attenuating amino acid,preferably lysine as E2 amino acid 314; codons at E2 amino acid position376 which specify an attenuating amino acid, preferably alanine as E2amino acid 376; codons at E2 amino acid position 372 which specify anattenuating amino acid, preferably leucine as E2 amino acid 372; codonsat nsP2 amino acid position 96 which specify an attenuating amino acid,preferably glycine as nsP2 amino acid 96; and codons at nsP2 amino acidposition 372 which specify an attenuating amino acid, preferably valineas nsP2 amino acid 372. Suitable attenuating mutations wherein otheralphaviruses are employed are known to those skilled in the art.

Attenuating mutations are introduced into the RNA by performingsite-directed mutagenesis on the cDNA which encodes the RNA, inaccordance with known procedures. See, e.g., Kunkel, Proc. Natl. Acad.Sci. USA 82:488 (1985), the disclosure of which is incorporated hereinby reference in its entirety. Alternatively, mutations are introducedinto the RNA by replacement of homologous restriction fragments in thecDNA which codes for the RNA, in accordance with known procedures, or incDNA copies using mutagenic polymerase chain reaction methods.

Optionally, the helper nucleic acids contain mutations that would belethal to the virus if incorporated into its native configuration. Forexample, the alphavirus capsid and glycoprotein genes of the VEE genomeare normally encoded in a single open-reading frame (ORF). Duringtranslation of this ORF, the capsid cleaves itself from the growingpolypeptide by virtue of an autoprotease activity. The protease activityis based on an active serine motif similar to that of chymotrypsin,which requires interaction of three distinct amino acid residues(serine, aspartate and histidine). In the VEE capsid gene, the serine,aspartate and histidine residues are located at amino acids 226, 174 and152, respectively. Mutagenesis of one or all of these residues willcompromise the protease activity of the capsid and result in non-viableviruses. Any number of mutations are possible at each residue, and soare referred to collectively as “m226, m174, or m152”, wherein the “m”designates “mutant.” The actual residue numbers are different for eachalphavirus but are determined from primary amino acid sequence of thestructural protein ORF. In the context of a two helper system, in whichthe capsid gene is provided on a helper nucleic acid separately from theglycoprotein gene, there is no requirement for autoprotease activity.However, if the autoprotease activity of the capsid protein is disabled,any recombination event that brought the glycoprotein gene into the sameORF as the capsid gene would result in a non-functional virus. This isbecause the capsid protein would be unable to cleave itself from thegrowing polypeptide (e.g., from the glycoprotein). Therefore,incorporation of such a mutation in the capsid gene ablates theautoprotease function but leaves the RNA packaging function of thecapsid protein unaltered, thereby reducing further the probability ofproducing a replication competent virus.

Also provided are compositions comprising a first helper nucleic acid asdescribed herein and a replicon. Optionally, the composition comprises asecond helper nucleic acid comprising at least one microRNA targetsequence of an endogenous, cellular microRNA and a nucleic acid encodingan alphavirus structural protein, wherein the microRNA target sequenceis located in the translated or untranslated region (UTR) of the nucleicacid encoding the alphavirus structural protein, and wherein the firstand second helper nucleic acids encode different alphavirus structuralproteins. For example, the first helper nucleic acid encodes at leastone but not all alphavirus structural proteins, and the second helpernucleic acid encodes at least one alphavirus structural protein notencoded by the first helper nucleic acid. The first and second helpernucleic acids comprise the same or different microRNA target sequences.Optionally, an alphavirus structural protein on the first helper nucleicacid is the alphavirus capsid protein and an alphavirus structuralprotein on the second helper nucleic acid is an alphavirus glycoproteinor vice versa. Optionally, the composition further comprises a packagingcell. Suitable packaging cells are discussed in more detail below. Asdiscussed above, optionally the replicon encodes a polypeptide, animmunogenic polypeptide, an immunostimulatory polypeptide or atherapeutic product.

Also provided herein are methods for the preparation of infective,propagation-defective, virus-like replicon particles in cell culture.Thus, provided is a method of making virus-like replicon particles (VRP)comprising (a) transfecting a cell with (i) a replicon, and (ii) one ormore helper nucleic acids as described herein, wherein the structuralproteins necessary to make the virus-like replicon particle are encodedby one or more of the cell, the replicon or the helper nucleic acid; andculturing the cell under conditions that allow for production ofassembled virus-like replicon particles comprising the replicon.Optionally, the replicon comprises a packaging signal. Optionally, thevirus-like replicon particles further comprise the helper nucleicacid(s) or a fragment thereof. As utilized herein, a fragment thereof isdefined as a portion of the helper nucleic acid that contains themicroRNA target sequence or sequences. By way of an example, thestructural proteins necessary to make a virus-like replicon particle areencoded by the helper nucleic acid(s). Optionally, the cell istransfected with a first helper nucleic acid and a second helper nucleicacid, wherein the first helper nucleic acid encodes at least one but notall the structural proteins necessary to make a virus-like repliconparticle and the second helper nucleic acid encodes at least one or morealphavirus structural proteins not encoded by the first helper nucleicacid. Multiple different nucleic acid molecules, e.g. the first andsecond helper nucleic acids and the replicon nucleic acid, can beco-introduced into the packaging cell. Optionally, all three moleculescan be RNA or DNA, or one or more molecules may be RNA and the othermolecules can be DNA. Optionally, an inhibitor is introduced into thecell culture, e.g., by electroporation or by lipid-based transfection,to inhibit the activity of the endogenous, cellular microRNA(s) duringpackaging of the replicon RNA. Optionally, the packaging cell isselected from those cells or cell lines which do not contain themicroRNAs that recognize the microRNA target sequences present on thehelper nucleic acids. Optionally, the replicon encodes the targets tothe endogenous, cellular microRNA (e.g., tissue-specific ordevelopment-stage specific microRNAs). Optionally, the method furthercomprises the step of isolating the VRPs. The virus-like repliconparticles are propagation defective and infective. In alphavirusreplicon particles (ARPs), an alphavirus vector or replicon, isoptionally engineered to contain and express one or more genes ofinterest. Alternatively, ARPs that do not express a gene of interest oran inhibitory molecule, sometimes referred to as an empty ARP, (see,e.g., WO2006/085983 to Johnston et al.) are used as adjuvants to enhancethe response to an immunogen, including another ARP. Thus, the repliconcan encode a polypeptide, an immunostimulatory molecule, an immunogenicpolypeptide, a therapeutic molecule, or nothing, as discussed above. Thealphavirus replicon vector can be derived from any alphavirus, such asVenezuelan Equine Encephalitis (VEE) virus, Sindbis virus, e.g. strainTR339, South African Arbovirus No. 86, and Semliki Forest virus, amongothers. The replicon is then introduced into cells in culture that arepermissive for the replication of alphaviruses and in which thestructural proteins of the alphavirus are also expressed, so that thereplicon is packaged by the structural proteins into ARPs. Methods forthe economical and efficient production of high yields of alphavirusreplicon particles are described in U.S. Pat. No. 7,078,218 to Smith etal., as are specific attenuated strains and viruses useful for theproduction of an ARP.

Provided herein are cells comprising one or more helper nucleic acids asdescribed herein and one or more replicons. The cell, also referred toas a helper cell or packaging cell, are used to produce infectious,propagation defective alphavirus particles. The cell must express or becapable of expressing alphavirus structural proteins sufficient topackage the replicon nucleic acid. The structural proteins are producedfrom one or more RNAs that are introduced into the helper cellconcomitantly with or prior to introduction of the replicon vector. SuchRNAs are optionally stably transformed into the packaging cell line.Thus, provided is a cell comprising a first helper nucleic acid asdescribed herein comprising at least one microRNA target sequence of anendogenous, cellular microRNA and a replicon. The cell optionallyfurther comprises an inhibitor of the endogenous, cellular microRNA.Such inhibitors, which are small RNA molecules, can be introduceddirectly into the packaging cell, concomitantly with the helper nucleicacid(s). As discussed above, optionally the replicon encodes apolypeptide, an immunogenic polypeptide or a therapeutic product.Optionally, the cell comprises a second helper nucleic acid comprisingat least one microRNA target sequence of an endogenous, cellularmicroRNA and a nucleic acid encoding an alphavirus structural protein,wherein the microRNA target sequence is located in the translated oruntranslated region (UTR) of the nucleic acid encoding the alphavirusstructural protein, and wherein the first and second helper nucleicacids encode different alphavirus structural proteins. The first andsecond helper nucleic acids comprise the same or different microRNAtarget sequences. Optionally, the alphavirus structural protein on thefirst helper nucleic acid is an alphavirus capsid protein and thealphavirus structural protein on the second helper nucleic acid is analphavirus glycoprotein or vice versa. By way of example, the firsthelper RNA includes RNA encoding at least one alphavirus structuralprotein but does not encode all alphavirus structural proteins.Optionally, the first helper RNA comprises RNA encoding the alphavirusE1 glycoprotein, but not encoding the alphavirus capsid protein and thealphavirus E2 glycoprotein. Alternatively, the first helper RNAcomprises RNA encoding the alphavirus E2 glycoprotein, but not encodingthe alphavirus capsid protein and the alphavirus E1 glycoprotein.Optionally, the first helper RNA comprises RNA encoding the alphavirusE1 glycoprotein and the alphavirus E2 glycoprotein, but not thealphavirus capsid protein. The first helper RNA optionally comprises RNAencoding the alphavirus capsid, but none of the alphavirusglycoproteins. As another example, the first helper RNA may comprise RNAencoding the capsid and one of the glycoproteins, i.e., either E1 or E2,but not both. In combination with any one of these first helper RNAs,the second helper RNA encodes at least one alphavirus structural proteinnot encoded by the first helper RNA. For example, where the first helperRNA encodes only the alphavirus E1 glycoprotein, the second helper RNAencodes one or both of the alphavirus capsid protein and the alphavirusE2 glycoprotein. Where the first helper RNA encodes only the alphaviruscapsid protein, the second helper RNA includes RNA encoding one or bothof the alphavirus glycoproteins. Where the first helper RNA encodes onlythe alphavirus E2 glycoprotein, the second helper RNA encodes one orboth of the alphavirus capsid protein and the alphavirus E1glycoprotein. Where the first helper RNA encodes both the capsid andalphavirus E1 glycoprotein, the second helper RNA includes RNA encodingone or both of the alphavirus capsid protein and the alphavirus E2glycoprotein.

The terms helper cell and packaging cell are used interchangeably hereinand refer to the cell in which alphavirus replicon particles areproduced. The helper cell comprises a set of helpers that encode one ormore alphavirus structural proteins. As disclosed herein, the helpersmay be RNA or DNA. The cell can be any cell that isalphavirus-permissive, i.e., cells that are capable of producingalphavirus particles upon introduction of a viral RNA transcript.Alphaviruses have a broad host range. Examples of suitable packagingcells include, but are not limited to, Vero cells, baby hamster kidney(BHK) cells, chicken embryo fibroblast cells, DF-1, 293, 293T, ChineseHamster Ovary (CHO) cells, and insect cells. The helper or packagingcell may optionally include a heterologous RNA-dependent RNA polymeraseand/or a sequence-specific protease. The nucleic acids encodingalphavirus structural proteins can be present in the helper celltransiently or by stable integration into the genome of the helper cell.The nucleic acid encoding the alphavirus structural proteins that areused to produce alphavirus particles can be under the control ofconstitutive and/or inducible promoters. For example, the alpha virusstructural protein coding sequences are provided on a single DNA helper(see, e.g., U.S. Pat. No. 7,045,335 to Smith et al.) or as two helperconstructs comprising an IRES element in which the translation of thesecoding sequences can be controlled by the activity of an IRES element.Optionally, the IRES element are active in the specific helper cell typeand not active, or minimally active in other cells types. Optionally,the helper(s) may comprise a subgenomic promoter-IRES-structural proteinnucleic acid cassette, in which the subgenomic promoter directstranscription of RNA and the IRES directs most or all expression of thestructural protein. The helper cells comprise nucleic acid sequencesencoding the alphavirus structural proteins in a combination and/oramount sufficient to produce an alphavirus particle when a recombinantreplicon nucleic acid is introduced into the cell under conditionswhereby the alphavirus structural proteins are produced and therecombinant replicon nucleic acid is packaged into alphavirus particle.

A promoter for directing transcription of RNA from DNA, i.e., a DNAdependent RNA polymerase, can be employed to produce the providednucleic acids. A promoter is a sequence of nucleotides recognized by apolymerase and sufficient to cause transcription of an associated(downstream) sequence. In the RNA helper systems and to produce thereplicon RNA, a promoter is utilized to synthesize RNA in an in vitrotranscription reaction, and specific promoters suitable for this useinclude the SP6, T7, and T3 RNA polymerase promoters. In the DNA helpersystems, the promoter functions within the packaging cell to directtranscription of messenger RNA encoding structural proteins necessaryfor packaging. Potential promoters for in vivo transcription of theconstruct include eukaryotic promoters such as RNA polymerase IIpromoters, RNA polymerase III promoters, or viral promoters such as MMTVand MoSV LTR, SV40 early region, RSV or CMV. Many other suitablemammalian and viral promoters are available. Alternatively, DNAdependent RNA polymerase promoters from bacteria or bacteriophage, e.g.,SP6, T7, and T3, may be employed for use in vivo, with the matching RNApolymerase being provided to the cell, either via a separate plasmid,RNA vector, or viral vector. Optionally, the matching RNA polymerase canbe stably transformed into a helper cell line under the control of aninducible promoter.

DNA constructs that function within a cell can function as autonomousplasmids transfected into the cell or they can be stably transformedinto the genome. The promoter is optionally a constitutive promoter,i.e., a promoter which, when introduced into a cell and operably linkedto a downstream sequence, directs transcription of the downstreamsequence upon introduction into the cell, without the need for theaddition of inducer molecules or a change to inducing conditions.Alternatively, the promoter may be regulated, i.e., not constitutivelyacting to cause transcription of the associate sequence. A regulatedpromoter can, for example be inducible. An inducible promoter acts sothat the cell only produces the functional messenger RNA encoded by theconstruct when the cell is exposed to the appropriate stimulus(inducer). An inducible promoter transcribes the associated sequenceonly when (i) an inducer molecule is present int eh medium in or onwhich the cells are cultivated, or (ii) conditions to which the cellsare exposed are changed to be inducing conditions. When using aninducible promoter, the helper constructs are introduced into thepackaging cell concomitantly with, prior to (either transiently orthrough stably transformation of the packaging cell line), or afterexposure to the inducer, and expression of the alphavirus structuralproteins occurs when both the constructs and the inducer are present.Alternatively, constructs designed to function within a cell can beintroduced into the cell via a viral vector, e.g., adenovirus, poxvirus,adeno-associated virus, SV40, retrovirus, nodavirus, picornavirus,vesicular stomatitis virus, and baculoviruses with mammalian pol IIpromoters.

Optionally, the RNA vector replicon is transcribed in vitro from a DNAplasmid and then introduced into the helper cell by electroporation.Alternatively, the RNA vector replicon is transcribed in vivo from a DNAvector plasmid that is transfected into the helper cell (see., e.g.,U.S. Pat. No. 5,814,482 to Dubensky et al.), or it is delivered to thehelper cell via a virus or virus-like particle. Once an RNA transcript(mRNA) encoding the helper or RNA replicon vectors is present in thehelper cell (either via in vitro or in vivo approaches), it iseventually translated to produce the encoded polypeptides or proteins.

Methods of inducing an immune response in a subject comprisingadministering to the subject a population of virus-like particles asdescribed herein or as made by the methods described herein are alsoprovided. Pharmaceutical compositions comprising an immunogenic amountof the infectious, propagation defective virus-like replicon particlesin combination with a pharmaceutically acceptable carrier areadministered to the subject. Exemplary pharmaceutically acceptablecarriers include, but are not limited to, sterile pyrogen-free water,sterile pyrogen-free physiological saline solution, sterile water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof, as well as stabilizers, e.g. HSA or other suitable proteins andreducing sugars. Subjects which may be administered immunogenic amountsof the infectious, replication defective alphavirus particles of thepresent invention include human and animal (e.g., dog, cat, cattle,horse, pigs, donkey, mouse, hamster, monkeys, guinea pigs, birds, eggs)subjects. Administration may be by any suitable means, such asintraperitoneal, intramuscular, intradermal, intranasal, intravaginal,intrarectal, subcutaneous or intravenous administration.

Immunogenic compositions comprising the VRPs (which direct theexpression of the sequence(s) of interest when the compositions areadministered to a human or animal) or adjuvant preparations comprisingVRPs (which do not express any nucleic acids encoding antigens orinhibitors) produced using the methods described herein are formulatedby any of the means known. Such compositions, are typically prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. Lyophilized preparations are also suitable.

The immunogenic (or otherwise biologically active) VRP-containingcompositions are administered in a manner compatible with the dosageformulation, and in such amount as is prophylactically and/ortherapeutically effective. As used herein, an immunogenic amount is anamount of the infectious virus-like particles which is sufficient toevoke an immune response in the subject to which the pharmaceuticalformulation is administered. The quantity to be administered, which isgenerally in the range of about 10² to about 10¹² infectious units permL in a dose, depends on the subject to be treated, the route by whichthe VRPs are administered, the immunogenicity of the expression product,the types of effector immune responses desired, and the degree ofprotection desired. Optionally, about 10⁶ to 10¹² infectious units, orVRPs per dose, are administered to the subject. Optionally, about 10¹⁰to 10¹² infectious units, or VRPs per dose, is administered to thesubject. Optionally, about 10⁶, 10⁷, or 10⁸ infectious units, or VRPsper dose, is administered to the subject. Precise amounts of the activeingredient required to be administered may depend on the judgment of thephysician, veterinarian or other health practitioner and may be peculiarto each individual, but such a determination is within the skill of sucha practitioner.

The pharmaceutical composition is given in a single dose or multipledose schedule. A multiple dose schedule is one in which a primary coursemay include 1 to 10 or more separate doses, followed by other dosesadministered at subsequent time intervals as required to maintain and orreinforce the immune response, e.g., weekly or at 1 to 4 months for asecond dose, and if needed, a subsequent dose(s) after severalmonths/years.

As discussed above, it is contemplated that other viral-based vectorsystems including, for example, retroviral (e.g. murine stem cellvirus), lentiviral, herpesviral and adenoviral vector systems, can bemodified as described herein for alphaviruses. In these systems, acritical function of the parental virus has been removed to set up areplication/propagation deficient vector system. The critical functionis provided in trans during production of the virus-like particles, andthe use of miRNA targets as described herein allows significantreduction in the probability of regeneration of functional,replication-competent virus in the recipient. As used herein, the phrasevector system refers to the components necessary to create virus-likeparticles. For example, a vector system comprises a vector, which ispackaged into the virus-like particles and one or more additionalnucleic acids that encode the genes necessary for production of thevirus-like particle. Optionally, the vector also comprises one or moregenes necessary for production of the virus-like particle. As usedherein, the critical function can be a gene necessary for production ofa virus-like particle such as a structural gene (i.e., a gene encodingan envelope protein such as a VSVG protein) or a gene necessary forreplication of the virus (i.e., a gene encoding a polymerase proteinsuch as Adenovirus E1a protein).

Thus, provided is a vector system comprising a helper nucleic acidcomprising (i) at least one microRNA target sequence of an endogenous,cellular microRNA and (ii) a nucleic acid encoding a viral structuralprotein, wherein the target sequence is located in the 3′ UTR, 5′ UTR ortranslated region of the viral structural protein, and a vector. Alsoprovided are vector systems comprising a helper nucleic acid comprising(i) at least one microRNA target sequence of an endogenous, cellularmicroRNA, and (ii) a nucleic acid encoding a viral protein essential forreplication of the virus, and a vector. The helper nucleic acid and/orvector is DNA or RNA. Optionally, the helper nucleic acid is located ina packaging cell. Optionally, the helper nucleic acid is located on aplasmid in the packaging cell. As discussed above for replicons,optionally the vector encodes a polypeptide, an immunostimulatorymolecule, an immunogenic polypeptide or a therapeutic product.

Optionally, the vector system is a single-cycle vector system or alimited-cycle vector system. As used herein, the term single-cyclevector system refers to a vector system that produces viral particlesthat are infectious but propagation-defective, i.e., the virus cannotpropagate beyond the host cell into which the particles are initiallyinfected. As used herein, the term limited-cycle vector system refers toa vector system that produces viral particles that are infectious andhave limited propagation capacity. For example, viral particles producedfrom limited-cycle vector systems can propagate beyond the host cellinto which the particles are initially infected, but are not virulent(i.e., the viral particles themselves are not pathogenic).

As discussed above, the microRNA target sequence of the helper nucleicacid is located, for example, in the translated region, the 5′ UTR, orthe 3′ UTR of the nucleic acid encoding the structural protein.Optionally, at least one target sequence is located in the 3′ UTR of thenucleic acid encoding a structural protein and at least one targetsequence is located in the 5′ UTR of the nucleic acid encoding astructural protein or viral protein essential for replication of thevirus. Preferably, the microRNA target sequence is located in the 3′UTRof the nucleic acid encoding the structural protein or viral proteinessential for replication and/or propagation of the virus.

Optionally, the provided vector systems further comprise a second helpernucleic acid comprising at least one microRNA target sequence of anendogenous, cellular microRNA and a nucleic acid encoding viralstructural protein(s) or encoding viral protein(s) essential forreplication and/or propagation of the virus. The first and second helpernucleic acids comprise the same or different microRNA target sequences.

Viral structural proteins include, but are not limited to lentivirus,herpesvirus, rhabdovirus, picornavirus, murine or feline leukemia virus,adenovirus and flavivirus structural proteins. Examples of lentivirusstructural proteins include human immunodeficiency virus (HIV)structural proteins. Examples of herpesvirus (HSV) structural proteinsinclude HSV-1 structural proteins. Examples of rhabdovirus structuralproteins include vesicular stomatitis virus (VSV) structural proteins.

Viral proteins essential for replication and/or propagation (e.g.,nonstructural proteins) are selected from the group consisting of alentivirus, herpesvirus, rhabdovirus, picornavirus, murine or felineleukemia virus, adenovirus and a flavivirus protein essential forreplication. Examples of lentivirus proteins essential for replicationproteins include human immunodeficiency virus (HIV) proteins essentialfor replication. Examples of herpesvirus (HSV) proteins essential forreplication include HSV-1 proteins essential for replication. Examplesof rhabdovirus proteins essential for replication include vesicularstomatitis virus (VSV) proteins essential for replication.

As an example, lentiviral vector packaging systems provide separatepackaging constructs for gag/pol and env, and typically employ aheterologous or functionally modified envelope protein for safetyreasons. See, e.g., Miller and Buttimore, Mol. Cell. Biol. 6(8):2895-902(1986). As used herein, the term packaging construct refers to aconstruct comprising a gene encoding a protein that is necessary forpackaging of the virus. This differs from the packaging signal, which islocated on the nucleic acid that is to be packaged into the virus. Thesemodifications minimize the homology between the packaging genome and theviral vector so that the ability of the vector to form recombinants isreduced (see, e.g., Miller and Rosman, BioTechniques 7(9):980-90(1989)). For other lentiviral vector systems, the accessory genes, vif,vpr, vpu and nef, are deleted or inactivated and the packaging functionsare divided into two genomes: one genome expresses the gag and pol geneproducts, and the other genome expresses the env gene product (see,e.g., Bosselman et al., Mol. Cell. Biol. 7(5):1797-806 (1987); Markowitzet al., J. Virol. 62(4):1120-4 (1988); Danos and Mulligan, Proc. Natl.Acad. Sci. USA 85:6460-6464 (1988)). Thus, microRNA targets can bepositioned in the translated or untranslated region of the nucleic acidsencoding these genes to prevent formation of replication competentvirus.

Flavivirus-based replicon systems have been described (Khromykh et al.,J. Virol. 72:5967-77 (1998); Scholle et al., J. Virol. 78:11605-14(2004); and Yoshii et al., Vaccine 23:3946-56 (2005)). Flavivirus-basedreplicon systems have the structural protein gene coding region deletedrendering the replicon vector incapable of producing new progeny incells that do not supply them in trans. As described for alphavirusvectors, introduction of miRNA target sequences into the 5′ and/or 3′UTR of the transcripts that provide the flavivirus structural proteinstargets those RNAs for destruction by cellular miRNAs when miRNAinhibitors are not present. If, during production of flavivirus repliconparticles in packaging cells, a recombination event restored thestructural protein coding region into the replicon vector, thesubsequent particle would still be propagation defective. This isbecause, when introduced into other cells, the structural protein RNAwould be targeted for degradation by the cellular miRNA degradationmachinery.

Propagation-deficient replicon systems based on Rhabdoviruses (e.g.,VSV), Paramyxoviruses (e.g., Sendai virus) and Picornaviruses (e.g.,Polio) require that structural proteins be provided in trans to packagevirus-like particles that can subsequently be used to infect targetcells (Matthias et al., Cell 90:849-57 (1997); Kapadia et al., Virology376:165-72 (2008); Ansardi et. al., J. Virol. 67:3684-90 (1993); andInoue et al., J. Gene. Med. 6:1069-81 (2004)). The helper function(e.g., the structural proteins) can be provided by plasmid DNAs, otherrecombinant vectors (e.g., vaccinia virus) or cells modified to expresshelper functions (e.g., structural proteins) either transiently orconstitutively. The helper structural protein mRNA transcripts canpotentially recombine into the replicon vector systems, thusreconstituting a propagation/replication competent viral genome.Engineering miRNA targets into the helper genes for these vector systemswould prevent formation of a propagation/replication competent viralgenome. This is because the structural protein mRNA is targeted fordegradation by the cellular miRNA degradation machinery.

Adenoviral vectors have been developed that are replication-defective.This has been accomplished by removal of the genes from the vector thatare essential for the virus to replicate. Replication-defectiveadenovirus vectors are produced in packaging cell lines that provide theelements required for replication and packaging of the defectiveadenovirus genome into virus-like particles. However, adenovirus vectorsmay become replication competent if DNA-DNA recombination events in thepackaging cell line occur. By engineering miRNA target sequences intothe genes essential for replication of the adenovirus, recombinantadenoviruses would not be replication competent since the mRNAs of thegenes essential for replication are targeted for degradation by themiRNA degradation machinery. In other words, if DNA recombination eventsoccur generating a full complement of adenovirus genes, the essentialreplication and packaging proteins would not be expressed due to miRNAtargeted degradation of those mRNA transcripts in subsequently infectedcells.

The same strategy described above will work for replication-incompetentor disabled infectious single cycle (DISC) herpes simplex virus type 1(HSV-1)-based vaccine vectors (Farrell et al., J. Virol. 68:927-32(1994); and Weir and Elkins, Proc. Natl. Acad. Sci. USA 90:9140-4(1993)). These HSV-1 vectors have deletions in their viral genomes ofessential genes (e.g., ICP4 gene and gH gene) that render themreplication or propagation-incompetent. HSV-1 virus-like particles canbe produced if the proteins encoded by the deleted portions of thegenome are provided in trans by cell lines that express them in either acontinuous or inducible manner. Placing miRNA targets into the essentialgenes expressed within the packaging cells would target those mRNAs fordegradation in subsequently infected cells. In other words, if DNA-DNArecombination occurs with the essential genes expressed by the packagingcell and the HSV-1 vectors, the essential genes would not be expressedin subsequently infected cells due to miRNA targeted degradation of theessential gene mRNA transcripts.

Further provided are kits comprising one or more of any of the helpernucleic acids described herein. As an example, the kit comprises ahelper nucleic acid comprising at least one microRNA target sequence ofan endogenous, cellular microRNA; and a nucleic acid encoding analphavirus structural protein, wherein the microRNA target sequence islocated in a region of the nucleic acid encoding the alphavirusstructural protein. For example, the microRNA target sequence can be inthe translated or untranslated region of the alphavirus structuralprotein. Optionally, the kits further comprise any of the repliconsdescribed herein. Optionally, the kits further comprise any of the cellsdescribed herein. Optionally, the kit comprises a population ofalphavirus-like replicon particles (ARPs). The ARPs can, for example,comprise a first subset of particles comprising a replicon and a secondsubset of particles comprising any of the helper nucleic acids orfragments thereof described herein and a replicon. Optionally, the kitsfurther comprise directions for making virus-like replicon particles.The directions can, for example, include any of the methods of makingvirus-like replicon particles described herein.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

EXAMPLES

Materials and Methods

Construction of helpers containing 3′ plus-strand microRNA (miRNA)target sequences. This set of helpers was designed to have the targetfor cellular miRNA action be present in the 3′ UTR region of thepositive-strand RNA message produced during helper replication. Thehelper design is schematically depicted in FIG. 1. A DNA fragment wassynthesized that coded for the reverse complement (RC) sequence of sixdifferent microRNAs (miRNA) aligned in tandem(5′-gcatgcaactatacaacctactacctcaacacagtcgaaggtctcagggacttcagttatcacagtactgtagatatccccctatcacgattagcattaaactacctgcactgtaagcactttgtcagttttgcatagatttgcacagtttaaac-3′(SEQ ID NO:17). The miRNA target sequences were specific for let-7 (RC1;5′-aactatacaacctactacctca-3′ (SEQ ID NO:11)), lin-4 (RC2;5′-acacagtcgaaggtctcaggga-3′ (SEQ ID NO:12)), miR-101(RC3;5′-cttcagttatcacagtactgta-3′ (SEQ ID NO:13)), miR155 (RC4;5′-cccctatcacgattagcattaa-3′ (SEQ ID NO:14)), miR-17 (RC5;5′-actacctgcactgtaagcactttg-3′ (SEQ ID NO:15)), and miR-19 (RC6;5′-tcagttttgcatagatttgcaca-3′ (SEQ ID NO:16)). A unique SphI restrictionsite was engineered before the 5′ most miRNA target sequence (let-7), aunique EcoRV restriction site was engineered between the third (miR-101)and fourth (miR-155) miRNA target sequences and a unique PmeIrestriction site was engineered after the last miRNA (miR-19) targetsequence. In addition, immediately downstream of the miRNA targetsequences 63 base pairs (bp) corresponding to the Venezuelan equineencephalitis (VEE) virus 3′ noncoding region (NCR), 55 bp correspondingto a poly (A) stretch and 8 bp corresponding to a unique NotIrestriction site were also synthesized. The 281 bp fragment was digestedwith SphI and NotI restriction enzymes and then ligated into capsid(dHcap6-mut1 (W-stop)) and GP (dHgp6-mut1) helper plasmids linearizedwith the same two enzymes. The resulting helper plasmids were designateddHcap6-mut1(W-stop)RC1-6 and dHgp6-mut1-RC1-6. The miRNA targets wererepresented by the identifiers 1 through 6 and are numbered in the orderlisted above (let-7=1, lin-4=2, miR-101=3, miR155=4, miR-17=5, andmiR-19=6).

Helper plasmids coding for the first three (let-7, lin-4 and miR-101;5′-gcatgcaactatacaacctactacctcaacacagtcgaaggtctcagggacttcagttatcacagtactgta-3′(SEQ ID NO:18)) or the last three (miR-155, miR-17 and miR-19;5′-cccctatcacgattagcattaaactacctgcactgtaagcactttgtcagttttgcatagatttgcacagtttaaac-3′(SEQ ID NO:19)) miRNA target sequences were constructed. Helpers withthe first three miRNA targets (1-3) were constructed by digestingdHcap6-mut1(W-stop)RC1-6 and dHgp6-mut1-RC1-6 helpers with EcoRV andNotI restriction enzymes to remove the miRNA targets 4-6 and the VEE 3′NCR. The VEE 3′ NCR was replaced by digesting dHcap6-mut1 (W-stop) withSphI, treating the DNA with T4 DNA polymerase to produce a blunt end andthen digesting the DNA further with NotI to release a 122 bp fragment.The 122 bp fragment was then ligated into the EcoRV/NotI digested capsidand gp helpers above, generating helpers designateddHcap6-mut1(W-stop)RC1-3 and dHgp6-mut1-RC1-3.

Helpers coding for the last three miRNA targets (4-6) were constructedby digesting dHcap6-mut1(W-stop)RC1-6 and dHgp6-mut1-RC1-6 helpers withEcoRV and RsrII restriction enzymes to remove the capsid or gp genes andthe miRNA targets 1-3. The capsid and gp genes were replaced bydigesting dHcap6-mut1 (W-stop) or dHgp6-mut1 DNA with SphI, treating theDNAs with T4 DNA polymerase to produce a blunt end and then digestingthe DNA further with RsrII to release the VEE structural protein genes.The RsrII/SphI(T4) capsid and gp gene fragments were gel purified andthen ligated into the RsrII/EcoRV digested miRNA 4-6 plasmid backbonesdescribed above, generating helpers designated dHcap6-mut1(W-stop)RC4-6and dHgp6-mut1-RC4-6.

Helpers coding for either individual miRNA targets or combinations oftwo miRNA targets were also constructed. A similar approach was used togenerate the miRNA target containing helpers as was used to produce the1-3 and 4-6 miRNA target helpers described above. PCR primers weredesigned that would amplify the individual miRNA targets as well as thetwo miRNA target combinations. The primers and DNA templates used toamplify the miRNA targets are summarized in Table 1. The miRNA targetswere cloned into the dHcap6-mut1 (W-stop) or dHgp6-mut1 helpers asdescribed above either as RsrII/PmeI fragments (miRNA targets 1, 1-2, 3,4, 4-5, and 5) or SphI/NotI fragments (miRNA targets 6 and 5-6). AllmiRNA helper constructs were sequenced to ensure that no errors wereintroduced during PCR amplification.

TABLE 1 Primers and DNA templates. miRNA miRNA PCR target Forward primerReverse primer template 1 T7 (5′- TTAATACGACTClet-7 RC (PmeI) R (5′-GGGGTT capsid or gp ACTATAG-3′ (SEQ ID TAAACTGAGGTAGTAGGTTGTATAGTT- miRNA 1-3 NO: 1)) 3′ (SEQ ID NO: 6)) helper1-2 T7 (SEQ ID NO: 1) lin-4 RC (PmeI) R (5′-GGGGTT capsid or gpTAAACTCCCTGAGACCTTCGACTGTGT- miRNA 1-3 3′ (SEQ ID NO: 7)) helper 3miR-101 RC (SphI) F 3-1.1pr1 (5′-TAAGAGCCGCGAGCG capsid or gp(5′-TTTGCATGCCTTCAGTT ATCCT-3′ (SEQ ID NO: 8)) miRNA 1-3ATCACAGTACTGTA-3′ helper (SEQ ID NO: 2)) 2-3 lin-4 RC (SphI) F3-1.1pr1 (SEQ ID NO: 8) capsid or gp (5′-TTTGCATGCACACAGTCGA miRNA 1-3AGGTCTCAGGGA-3′ helper (SEQ ID NO: 3)) 2 T7 (SEQ ID NO: 1)lin-4 RC (PmeI) R capsid or gp (SEQ ID NO: 7) miRNA 2-3 helper 4T7 (SEQ ID NO: 1) miR-155 RC (PmeI) R (5′-GGGTT capsid or gpTAAACTTAATGCTAATCGTGATAGGGG- miRNA 4-6 3′ (SEQ ID NO: 9)) helper 4-5T7 (SEQ ID NO: 1) miR-17 RC (PmeI) R (5′-GGGT capsid or gpTTAAACCAAAGTGCTTACAGTGCAGGT miRNA 4-6 AGT-3′ (SEQ ID NO: 10)) helper 5T7 (SEQ ID NO: 1) miR-17 RC (PmeI) R capsid or gp (SEQ ID NO: 10)miRNA 5-6 helper 5-6 miR-17 RC (SphI) F 3-1.1pr1 (SEQ ID NO: 8)capsid or gp (5′-CATGCATGCACTACCTGCA miRNA 4-6 CTGTAAGCACTTTG-3′ helper(SEQ ID NO: 4)) 6 miR-19 RC (SphI) F 3-1.1pr1 (SEQ ID NO: 8)capsid or gp (5′-CATGCATGCTCAGTTTTGC miRNA 4-6 ATAGATTTGCACA-3′ helper(SEQ ID NO: 5))

Helpers coding for individual miRNA targets repeated six times, on thepositive-strand RNA message produced during helper replication, werealso constructed. Six copies of each individual miRNA target were chosenbecause that is the total number of miRNA targets tested in the RC1-6constructs and the length of the inserted sequence in the 3′ NCR wouldalso be maintained in the new constructs relative to the RC1-6constructs. DNA fragments were de-novo synthesized (BlueHeronBiotechnology, Inc.; Bothell, Wash.) that coded for the RC sequence ofsix copies of each individual miRNA aligned in tandem. The identifiersfor the respective 6mer miRNA fragments were: RC1x6, RC2x6, RC3x6,RC4x6, RC5x6 and RC6x6. A unique SphI restriction site was engineeredbefore the first miRNA target sequence, a unique EcoRV restriction sitewas engineered between the third and fourth copy of the miRNA targetsequence and a unique PmeI restriction site was engineered after thelast copy of the miRNA target sequence. The respective miRNAx6 sequencefragments were digested with SphI and PmeI restriction enzymes and thenligated individually in place of the miRNA1-6 sequence fragment found indHcap6-mut1(W-stop)RC1-6 or dHgp6-mut1-RC1-6, by digesting the helperswith SphI and PmeI restriction enzymes. The resulting capsid helperswere designated dHcap6-mut1(W-stop)RC1x6, dHcap6-mut1(W-stop)RC2x6,dHcap6-mut1(W-stop)RC3x6, dHcap6-mut1(W-stop)RC4x6,dHcap6-mut1(W-stop)RC5x6 and dHcap6-mut1(W-stop)RC6x6. The resulting GPhelper were designated dHgp6-mut1-RC1x6, dHgp6-mut1-RC2x6,dHgp6-mut1-RC3x6, dHgp6-mut1-RC4x6, dHgp6-mut1-RC5x6 anddHgp6-mut1-RC6x6.

Helpers coding for three copies of a miRNA target sequence (e.g., RC1x3)were also constructed. This was accomplished in two ways. For miRNAtargets RC1-RC6, helpers containing three copies were produced bydigesting the capsid and gp helpers that contained six copies of anindividual miRNA target (e.g., RC1x6) with restriction enzymes EcoRV andPmeI. This digestion releases a 75 bp fragment removing 3 of the 6 miRNAtarget copies from each helper. The EcoRV/PmeI digested DNAs were thenrelegated on themselves generating capsid and gp helpers with 3 miRNAtargets in their 3′ UTRs. Six additional miRNA target sequences wereidentified (RC7-RC12) that were known to have more cell-type ortissue-specific activity than the more broadly active targetsrepresented by RC1-RC6. The miRNA targets for RC7-RC12 are as follows:RC7=miR-143 (5′-gagctacagtgcttcatctca-3′ (SEQ ID NO:20)); RC8=miR-30b(5′-agctgagtgtaggatgtttaca-3′ (SEQ ID NO:21)); RC9=miR-181(5′-actcaccgacagcgttgaatgtt-3′ (SEQ ID NO:22)); RC10=miR-124(5′-ggcattcaccgcgtgcctta-3′ (SEQ ID NO:23)); RC11=miR-1(5′-atacatacttctttacattcca-3′ (SEQ ID NO:24)); and RC12=miR-133a(5′-cagctggttgaaggggaccaaa-3′ (SEQ ID NO:25)). Capsid helpers thatcontained target sequences consisting of three copies of each specificmiRNA target sequence were produced in the following manner. Reverse PCRprimers that coded for each miRNA target sequence in triplicate (e.g.,RC7x3 R(5′-GCGTTTAAACTGAGATGAAGCACTGTAGCTCTGAGATGAAGCACTGTAGCTCTGAGATGAAGCACTGTAGCTCGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:26)); RC8x3 R(5′-GCGTTTAAACTGTAAACATCCTACACTCAGCTTGTAAACATCCTACACTCAGCTTGTAAACATCCTACACTCAGCTGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:27)); RC9x3 R(5′-GCGTTTAAACAACATTCAACGCTGTCGGTGAGTAACATTCAACGCTGTCGGTGAGTAACATTCAACGCTGTCGGTGAGTGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:28)); RC10x3 R(5′-GCGTTTAAACTAAGGCACGCGGTGAATGCCTAAGGCACGCGGTGAATGCCTAAGGCACGCGGTGAATGCCGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:29)); RC11x3 R(5′-GCGTTTAAACTGGAATGTAAAGAAGTATGTATTGGAATGTAAAGAAGTATGTATTGGAATGTAAAGAAGTATGTATGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:30)); and RC12x3 R(5′-GCGTTTAAACTTTGGTCCCCTTCAACCAGCTGTTTGGTCCCCTTCAACCAGCTGTTTGGTCCCCTTCAACCAGCTGGCATGCTTACCATTGCTCGCAGTTCTCCGGAGTATACTTCACGGTAACTCCC-3′ (SEQ ID NO:31))) and a region ofhomologous capsid helper sequence were engineered. These reverse primerswere then combined with a capsid gene specific forward primer (capsid(RsrII) F; 5′-CCTCGGACCGACCATGTTCCCGTTCCAGCCAATG-3′ (SEQ ID NO:32)) toamplify a PCR product that coded for the entire capsid gene, threecopies of each miRNA target sequence (e.g., RC7x3) and the VEE 3′ UTRsequence. These capsid-RCx3 PCR products were then digested with RsrIIand PmeI restriction enzymes and used to replace the RsrII/PmeI regionof dHcap6-mut1(W-stop)RC1-6 DNA, generating dHcap6-mut1(W-stop)RC7x3,dHcap6-mut1(W-stop)RC8x3, dHcap6-mut1(W-stop)RC9x3,dHcap6-mut1(W-stop)RC10x3, dHcap6-mut1(W-stop)RC11x3 anddHcap6-mut1(W-stop)RC12x3. The entire capsid and miRNA target sequenceregions were sequenced to ensure no errors were introduced during PCRamplification. The RC7-RC12 target sequences were engineered with aunique 5′ SphI restriction site and a unique 3′ NotI restriction site.Each of the miRNA targeted capsid helper DNAs (RC7x3-RC12x3) weredigested with SphI and NotI restriction enzymes to release the ˜190 bptrimer miRNA target sequences. Each ˜190 bp trimer miRNA target(SphI/Not) fragment was then ligated into dHgp6-mut1 DNA linearized withSphI and NotI restriction enzymes to produce RC7x3-RC12x3 miRNA targetedGP helper constructs: dHgp6-mut1-RC7x3; dHgp6-mut1-RC8x3;dHgp6-mut1-RC9x3; dHgp6-mut1-RC10x3; dHgp6-mut1-RC11x3; anddHgp6-mut1-RC12x3.

Construction of helpers containing 3′ minus-strand miRNA targetsequences. Additional helpers were constructed that provided the targetsequences for cellular miRNA action on the minus-strand replicativeintermediate RNA. A forward primer specific to the miR-19 (miR-19 (SphI)F (SEQ ID NO:5)) sequence was engineered to code for a unique SphIrestriction site. A reverse primer specific for let-7 (let-7 RC (PmeI) R(SEQ ID NO:6)) was engineered with a unique PmeI restriction site. PCRamplification with these primers resulted in a 155 bp product. The PCRproduct was digested with SphI and PmeI restriction enzymes and ligatedinto SphI and PmeI linearized dHcap6-mut1(W-stop)RC1-6 anddHgp6-mut1-RC1-6 DNA. The resultant helpers (dHcap6-mut1(W-stop)RC1-6-NSand dHgp6-mut1-RC1-6-NS) generated the miRNA target sequences on theminus-strand replicative intermediate RNA.

Construction of helpers containing 5′ plus and minus-strand miRNA targetsequences. Helpers were constructed that contain the miRNA targetsequences (1-6) in the 5′ non-coding region (NCR) of these RNAs suchthat the targets would be present on either the plus or minus-strand RNA(FIG. 2). A unique RsrII restriction site was present immediately 5′ tothe structural gene in the dHcap6-mut1 (W-stop) helper plasmid and itwas used to generate these constructs. Forward (let-7 RC (RsrII) F(5′-TTTCGGTCCGAACTATACAACCTAC-3′ (SEQ ID NO:34))) and reverse (miR-19 RC(RsrII) R (5′-TTTCGGACCGTGTGCAAATCTATGC-3′) (SEQ ID NO:33)) primersengineered to code for RsrII restriction sites were designed to amplifythe miRNA 1-6 cassette. The 155 bp PCR product was digested with RsrIIrestriction enzyme and ligated into dHcap6-mut1 (W-stop) DNA linearizedwith RsrII enzyme. The orientation of the miRNA 1-6 PCR product wasdetermined by sequence analysis and clones with the miRNA 1-6 fragmentin only the reverse orientation (minus-strand RNA) were obtained. Theseclones were designated, dHcap6-mut1(W-stop)-5′RC1-6-NS. To generatehelpers with the miRNA 1-6 targets 5′ of the structural genes on theplus-stranded RNA additional steps were required. First, the 5′ RsrIIrestriction site introduced when the minus-strand construct was producedwas mutated to a unique BglII restriction site (primer: RsrII→Bgl IImut; (5′-GACATTGGAAGATCTTGTGCAAATCTATG-3′ (SEQ ID NO:35))) using sitedirected mutagenesis (Stratagene; Lo Jolla, Calif.), resulting in ahelper designated, dHcap6-mut1(W-stop)-5′BglII-RC1-6-NS. Next, primerswere engineered to amplify the plus-strand miRNA 1-6 target sequencewith a 5′ BglII restriction site and a 3′ RsrII restriction site (let-7RC (Bgl II) F (5′-GGAGATCTCGAACTATACAACCTAC-3′ (SEQ ID NO:36)); andmiR-19 RC (RsrII) R (SEQ ID NO:33)). The 147 bp product of amplificationwas digested with BglII and RsrII restriction enzymes and gel purified.The dHcap6-mut1(W-stop)-5′BglII-RC1-6-NS DNA was then digested withBglII and RsrII to remove the minus-stand oriented miRNA targets and theplus-strand miRNA target fragment cloned in its place. The helper thatexpressed the miRNA targets on the plus-strand RNA was designateddHcap6-mut1(W-stop)-5′RC1-6.

Construction of helpers containing 5′ and 3′ plus-strand miRNA targetsequences. A capsid helper was constructed that coded for miRNA1-6targets in both the 5′ and 3′ UTR positions on the plus-strand RNAtemplate (FIG. 3). To accomplish this the dHcap6-mut1(W-stop)-5′RC1-6helper DNA was digested with SphI and NotI restriction enzymes torelease the wild type 3′ UTR sequence. The dHcap6-mut1(W-stop)RC1-6helper DNA was digested with SphI and NotI restriction enzymes and the3′ UTR fragment (ca 270 bp) containing the miRNA1-6 targets was gelpurified. The linearized dHcap6-mut1(W-stop)-5′RC1-6 DNA was ligatedwith the 3′ UTR miRNA1-6 fragment and the resulting helper wasdesignated, dHcap6-mut1(W-stop)-5′ & 3′RC1-6 DNA.

Construction of miRNA targeted, cleavage deficient, capsid helpers. Sitedirected mutagenesis was used to introduce a glycine (G) residue inplace of the histidine (H) residue at amino acid 152 (m152) of thecapsid protein in miRNA targeted capsid helper constructs (primers:Cap152 G F (5′-GTTATTCAGGCCGATGGGTGTGGAAGGCAAGATCG-3′ (SEQ ID NO:37));and Cap152 G R (5′-CGATCTTGCCTTCCACACCCATCGGCCTGAATAAC-3′ (SEQ IDNO:38))). The m152 capsid gene was sequenced to ensure that no othermutations were introduced during mutagenesis. In order to demonstratethat the m152 H→G change ablates the autoprotease function of the capsidprotein, the m152 mutation was introduced into the capsid codingsequence of pHCMV-Vsp (see U.S. Pat. No. 7,045,335 to Smith et al.). ThepHCMV-Vsp DNA codes for a Cytomegalovirus (CMV) immediate early promoterthat controls the DNA-dependent RNA transcription of the complete VEEstructural protein coding region (capsid-E3-E2-6K-E1). Site directedmutagenesis was used to introduce the m152 mutation into the capsidprotein of the pHCMV-Vsp construct, generating pHCMV-Vsp(m152). Theentire VEE structural gene coding region was sequenced inpHCMV-Vsp(m152) to ensure that no other mutations were introduced duringmutagenesis.

Construction of helpers containing 9 miRNA target sequences. Helperswere constructed that contain one copy of 9 different miRNA targetsequences in the 3′ non-coding region (NCR) such that the targets wouldbe present on the plus strand RNA. The following miRNA targets werecombined on the capsid and glycoprotein helpers: RC1 (Let-7), RC4(miR-101), RC5 (miR-17), RC9(miR-181), RC2 (miR-125), RC3 (miR-101),RC10 (miR-124), RC7 (miR-143) and RC12 (miR-133a). DNA coding for the 9miRNA targets was de-novo synthesized with unique SphI and PmeIrestriction sites engineered at the 5′ and 3′ ends, respectively. UniqueSphI and PmeI restriction sites present immediately 3′ to the structuralgene in both helper plasmids were used to generate these constructs. Thehelper plasmids were linearized with SphI and PmeI and the approximately200 bp de novo synthesized 9 miRNA target fragment was cloned in atthese sites. These 9 miRNA target-containing helper plasmids wereidentified as (dHcap6-mut1(W-stop)mi and dHgp6-mut1 mi, respectively):In addition, the ˜200 bp 9 miRNA target fragment was cloned into thecapsid helper modified to code for the 152 capsid cleavage mutationresulting in generation of the helper dHcap6-mut1(W-stop)mi152.

Construction of miRNA-targeted replicon vectors. Replicon vectorsexpressing the CAT reporter gene were constructed that contained miRNAtargets located in the 3′ UTR. The miRNA targets were cloned into a CATreplicon vector as tandem single copies (RC1-6), as six tandem copies ofthe same miRNA target (RC1x6, RC2x6, RC3x6, RC4x6, RC5x6 and RC6x6) oras three tandem copies of the same miRNA target (RC7x3, RC8x3, RC9x3,RC10x3, RC11x3 and RC12x3). The replicon vector, pERK/342/EMCV/CAT, usedfor all constructions has been described previously (Kamrud et al.,Virology 360(2):376-87 (2007)). The miRNA target sequences forRC1x6-RC6x6 and RC7x3-RC12x3 were digested from their respectivedHcap6-mut1(W-stop)RC helpers (described above) using restrictionenzymes SphI and NotI. The miRNA target fragments released afterdigestion were then isolated and ligated into the pERK/342/EMCV/CATreplicon vector linearized with the same restriction enzymes. Theresulting replicon vectors were designated pERK/342/EMCV/CAT-RC1-6,pERK/342/EMCV/CAT-RC1x6, pERK/342/EMCV/CAT-RC2x6,pERK/342/EMCV/CAT-RC3x6, pERK/342/EMCV/CAT-RC4x6,pERK/342/EMCV/CAT-RC5x6, pERK/342/EMCV/CAT-RC6x6,pERK/342/EMCV/CAT-RC7x3, pERK/342/EMCV/CAT-RC8x3,pERK/342/EMCV/CAT-RC9x3, pERK/342/EMCV/CAT-RC10x3,pERK/342/EMCV/CAT-RC11x3 and pERK/342/EMCV/CAT-RC12x3.

A replicon vector expressing the influenza A/Wisconsin HA gene,pERK/342/EV71/A(Wis)/HA, was produced as described previously (Kamrud etal., Virology 360(2):376-87 (2007)). The RC1-6 miRNA sequence was clonedinto the 3′ UTR of the pERK/342/EV71/A(Wis)/HA replicon as describedabove for the miRNA targeted CAT replicons, generating a replicondesignated pERK/342/EV71/A(Wis)/HA-RC1-6.

RNA transcription, electroporation and Venezuelan equine encephalitisvirus replicon particle (VEE RP) production. Capped replicon and helperRNAs were in-vitro transcribed using a T7 RiboMax™ kit (Promega; MadisonWis.) following the manufactures instructions. RNAs were purified usingRNeasy™ purification columns (Qiagen; Valencia, Calif.) following themanufacturer's instructions. The procedures used for producing VEE RPvaccines were based on modifications of published methods (Pushko etal., Virology 239(2):389-401 (1997)). Vero cells (8×10⁷) suspended in0.6 ml PBS (GIBCO) were electroporated with 30 μg of replicon RNA, 20 μgof capsid helper RNA and 60 μg of GP helper RNA using a Bio-Rad GenePulser (Bio-Rad; Hercules, Calif.). When miRNA-targeted helpers orreplicons were used to generate VEE RP, miRNA inhibitors were includedin the electroporation. The miRNA inhibitors were 2′-O-methyl modifiedRNA oligonucleotides with the reverse complement (RC) sequence to thetarget miRNA (Eurogentec; San Diego Calif.). Cells were pulsed fourtimes with the electroporator set at 580 volts and 25 μF. Electroporatedcell suspensions were seeded into roller bottles containing serum-freemedium and incubated at 37° C. in 5% CO₂ for 16-24 hours. VEE RP wereharvested from culture fluids and the infectious titer of the VEE RPpreparation was measured by antigen-specific IFA and tested in acytopathic effect (CPE) assay to assure the absence of detectablereplication-competent virus. VEE RP generated with a replicon vectorcontaining miRNA-targets were titrated on Vero cells electroporated withmiRNA inhibitors to allow the replicon vector to replicate unhindered bythe cellular miRNAs. VEE RP were formulated with human serum albumin(1%) and stored at −80° C. until used.

IFA and CAT ELISA. CAT expression was quantified by ELISA usingmiRNA-targeted, CAT replicon VEE RP infected cell lysates and acommercially available CAT ELISA kit (Boehringer Mannheim; Indianapolis,Ind.) following the manufacturer's instructions. All cell types analyzedwere infected with VEE RP at a multiplicity of infection (MOI)=1 basedon genome copies rather than infectious units (IU). The cell lysateswere produced 18 hours post infection and total protein concentrationwas determined for each sample using a BCA protein kit (Pierce;Rockford, Ill.). CAT expression was also detected by immunofluorescenceassay (IFA) using a rabbit anti-CAT antibody (Cortex Biochem Inc.; SanLeandro, Calif.). Cells transfected with replicon RNA and helper RNAswere also analyzed by IFA using goat anti-nsP2 antibody (for replicon),goat anti-capsid antibody and goat anti-GP antibody. All cells for IFAanalysis were fixed with methanol and inspected using a Nikon EclipseTE300 ultraviolet fluorescence microscope.

Northern analysis. Northern analysis was carried out on total cellularRNA collected from VEE RP infected cells. Total cellular RNA wascollected from the cells 16 hours post infection using SV Total RNAIsolation columns (Promega) following the manufacturers instructions.The RNA was quantified by UV absorption and 3 μg of each were run on 1%glyoxal agarose gels before being transferred to a BrightStar-Plusmembrane (Ambion; Austin, Tex.) by passive transfer. The RNA was UVcrosslinked to the membrane, blocked with UltraHyb (Ambion) solution for1 hour at 45° C., and probed overnight with UltraHyb solution containingbiotinylated RNA probes specific for nsP2, capsid or GP genes at 65° C.After overnight hybridization the blot was processed forchemiluminescent RNA detection using a BrightStar BioDetect kit (Ambion)following the manufacturer's instructions and visualization afterexposure to autoradiograph film (Kodak; Rochester N.Y.).

Quantitative reverse transcription PCR(RTqPCR) analysis of VEE RP. Todetermine the number of genome equivalents present in eachmiRNA-targeted CAT replicon VEE RP preparation, a standard one-stepRT-qPCR protocol was performed on an Applied Biosystems 7500 Fast RealTime PCR System sequence detection system (Applied Biosystems; FosterCity, Calif.). Amplification was detected by means of a fluorogenicprobe designed to anneal to a region of the nsP2 gene on the repliconbetween the two primers. A 5′ reporter dye (6-FAM) and a 3′ quencher dye(BHQ-1) were attached to the probe. Proximity of the reporter andquencher dyes resulted in the suppression of reporter fluorescence priorto amplification. Upon successful amplification of the target region,the 5′ exonuclease activity of DNA polymerase released the reporter dyefrom the hybridized probe, resulting in a fluorescent signal. PurifiedVEE replicon RNA was used to generate a standard curve in the assay, andthe fluorescent signal of each VEE RP sample was measured up to fortyPCR cycles and compared to the fluorescent signal of the standards todetermine genome equivalents.

Vaccination of mice and sample collection. Mouse study 1: Female, 6-8week old, BALB/c, mice (Charles River Laboratory; Kingston, N.Y.) wereimmunized with 1×10⁶ IU of VEE RP expressing the influenza A/WisconsinHA gene that were produced using combinations of miRNA-targeted helpersand replicon vectors (Table 5). Mice were immunized at 0 and 3 weeks bysubcutaneous (SC) injection into the rear footpad. Influenza HA-specificantibody and T cell responses were monitored 1 week after eachimmunization. Blood was collected by retro-orbital bleeds for allgroups. Eight (8) mice from each group were sacrificed 1 week after eachimmunization and splenocytes collected from individual animals forT-cell analysis.

Mouse study 2: Groups of six (6), female, 6-8 week old, BALB/c, mice(Charles River Laboratory) were immunized with 5×10⁶ IU of VEE RPexpressing the herpes simplex virus II gD gene that were produced usingcombinations of miRNA-targeted helpers and replicon vectors (Table 6).Mice were immunized at 0 and 3 weeks by subcutaneous (SC) injection intothe rear footpad. Blood was collected by retro-orbital bleeds for allgroups one week after the second vaccination.

VEE RP Neutralization Assay. Serum used to supplement media and allmouse serum samples tested were heat inactivated at 56° C. for 30minutes. Vero cells were seeded into 96-well cell culture plates andincubate at 37° C., 5% CO₂ overnight. Serum samples were diluted 1:10 in96-well Titertek tubes with EMEM containing 1×NEAA, 1×PenStrepFungizone, and 5% heat inactivated FBS. Samples were further dilutedserially 1:2 in EMEM to reach neutralization endpoints. An equal volumeof diluted GFP VEE RP was added to each of the serum dilution wells andincubated at 4° C. overnight. After overnight incubation, 100 μL of eachsera/GFP VEE RP mixture was transferred to wells of Vero cells in96-well plates. The plates were incubated at 37° C., 5% CO₂ for 1 hourand then the sera/VEE RP mixture was removed from each well and replacedwith 100 μL fresh EMEM media. The plates were then incubate at 37° C.,5% CO₂ overnight. After overnight incubation the media was removed fromthe plates and each plate was rinsed with PBS 1 time. The number of GFPpositive cells was counted for control wells to determine an 80% cut-offvalue. The last dilution for each serum sample where the GFP positivecell count was less than 80% of the cut-off value was considered the 80%VEE RP neutralization titer.

HA ELISA. For HA ELISA analysis, dilutions of sera from VEE RPvaccinated mice were made into PBS containing 1% BSA and 0.05% Tween-20beginning with 1:40 followed by two fold dilutions out to 1:2560.Pre-bleed samples were diluted to only 1:40. ELISA plates (Nunc;Rochester, N.Y.), which had been coated with recombinant A/Wisconsin HAantigen (50 ng/well) (Protein Sciences Inc, Meriden, Conn.) incarbonate/bicarbonate buffer (Sigma-Aldrich; St. Louis, Mo.) overnightat 4° C., were incubated with 200 μl/well of blocking buffer (PBS, 3%BSA) at 30° C. for 1 hour. Blocked plates were washed three times with200 μl of PBS. Fifty μl of diluted sera was added to the plates induplicate and incubated at 30° C. for one hour. Plates were washed threetimes with 200 μl PBS. 100 μl of alkaline phosphatase-conjugatedanti-mouse polyvalent immunoglobulins (IgG, IgA, IgM) (Sigma) diluted inblocking buffer (1:500) was added to each well. Plates with secondaryantibody were incubated for 1 hour at room temperature and then washedthree times with 200 μl PBS. Plates were developed using Fast™p-Nitrophenyl phosphate tablet sets (Sigma) and reading at a wavelengthof 405. An OD₄₀₅ of 0.2 or greater was considered positive.

HA ELISPOT. Splenocytes were isolated from individual animals and HAspecific gamma interferon enzyme-linked immunospot (ELISPOT) assays wereperformed to determine the number of antigen-specific cytokine-secretingT cells. This procedure has been described previously (Reap et al.,Vaccine 25(42):7441-9 (2007)).

Results

Experiments to demonstrate functional miRNA activity on helper RNAs.Experiments were conducted to determine whether eukaryotic miRNAs couldbe used to control/reduce alphavirus helper replication/expression,within cells, by placing miRNA target sequences in the 3′ UTR of theseRNAs. The target sequences selected are representative of evolutionarilyconserved or ubiquitous eukaryotic miRNAs (Sempere et al., Genome Biol.5(3):R13 (2004); Baskerville and Bartel, RNA 11(3):241-7 (2005); Farh etal., Science 310(5755):1817-21 (2005)).

The initial experiment was designed to determine whether the RC1-6 miRNAtargets (let-7, lin-4, miR101, miR-155, miR-17, miR-19=targets RC1-6) orsubsets of these targets (let-7, lin-4, miR101=targets RC1-3 or miR-155,miR17, miR-19=targets RC4-6) would be able to control helper replicationwhen located in the 3′ UTR of the plus-strand RNA. Vero cells wereelectroporated with a replicon vector combined with capsid and gphelpers with matching complements of miRNA targets on them.Specifically, replicon RNA was combined with capsid and gp helpers withmiRNA targets RC1-6 on them or with both helpers carrying the miRNAtargets RC1-3 on them or with both helpers carrying the miRNA targetsRC4-6 on them. Each of the helper and replicon combinations wereelectroporated in the presence and absence of miRNA inhibitors(2′-O-methylated RNA oligonucleotides) specific for the completecomplement of miRNA targets present on the helpers. In addition, thehelper RNA combinations with miRNA targets RC1-3 and RC4-6 were alsocombined with unmatched miRNA inhibitor combinations to demonstrate thespecificity of the inhibitors used. For example, miRNA RC1-3 targetedhelper RNAs were treated with miRNA inhibitors specific for miRNAtargets RC4-6. The electroporated cells were seeded into media andincubated overnight. After incubation (˜18 hours), samples werecollected to examine helper-specific protein expression (Western blotand IFA), helper-specific RNA replication (Northern blot) and VEE RPyield. The results of capsid and gp Western blot analysis are shown inFIG. 4. The results of capsid and gp-specific Northern blot analysis areshown in FIG. 5. The VEE RP yields produced by the different miRNAtargeted helper RNA combinations is shown in FIG. 6. Western blotanalysis indicated that expression of capsid and GP was reduced in theabsence of miRNA inhibitors specific for the miRNA targets present onthe helpers (FIG. 4). The difference in protein expression wasparticularly evident for GP. Northern blot analysis indicated thatcapsid and GP helper replication levels mirrored the respective helperprotein expression in the presence or absence of the matched miRNAinhibitors (FIG. 5). The relative capsid and GP expression and helperRNA replication levels predicted the VEE RP yields produced. Those cellsthat did not receive the appropriate miRNA inhibitors (specific for themiRNA targets on the helpers) produced 3 to 4.5 orders of magnitude lessVEE RP (FIG. 6).

The experiment described above matched the miRNA targets on each helper.Next, an experiment was conducted to determine whether differentcombinations of miRNA targets could be present on the helpers and stillrescue VEE RP yields when the appropriate combination of miRNAinhibitors was supplied. Combinations of a capsid helper with RC1-3targets and a GP helper with RC4-6 (and vice-versa) were tested in thepresence or absence of a mixture of all 6 miRNA inhibitors. Western blotanalysis indicated that expression of capsid and GP was reduced in theabsence of miRNA inhibitors specific for the miRNA targets present onthe helpers (FIG. 7). The difference in protein expression, in theabsence of inhibitor, was more pronounced than that noted when the miRNAtargets present on the helpers were matched (FIG. 4). Northern blotanalysis indicated that capsid and GP helper replication levels mirroredthe respective helper protein expression in the presence or absence ofthe matched miRNA inhibitors (FIG. 8). As found in the experiment withmatched miRNA targeted helpers, the relative capsid and GP expressionand helper RNA replication levels predicted the VEE RP yields produced.VEE RP yields were 3 orders of magnitude lower in cells that did notreceive the miRNA inhibitors when compared to those that did (FIG. 9).

Experiments to determine location requirement of miRNA targets on helperRNAs. To determine whether the miRNA targets RC1-6 could function inlocations other than the 3′ UTR of the plus-strand capsid helper RNA,additional capsid helpers were constructed that moved the miRNA RC1-6targets to the 5′ UTR of the capsid helper RNA on both the plus-strandand minus-strand RNA templates for replication. In addition, the miRNARC1-6 targets were engineered in the 3′ UTR on the minus-strand RNAtemplate. These experiments were conducted with a combination ofreplicon RNA, unmodified GP helper RNA and one of the miRNA RC1-6targeted capsid helper RNAs in the presence and absence of miRNAinhibitors specific for the miRNA targets present on the helpers. Theelectroporated cells were seeded into media and incubated overnight.After incubation (˜18 hours), samples were collected to examine capsidhelper-specific protein expression (Western blot and IFA), RNAreplication (Northern blot) and VEE RP yield. The results ofcapsid-specific Western and Northern blot analysis are shown in FIG. 10and FIG. 11, respectively. The VEE RP yields produced using thedifferent miRNA targeted capsid helper is shown in FIG. 12. Both Westernand Northern blot analysis indicated that the miRNA targets functionmost effectively when on the plus-strand helper RNA. Helper expressionand replication were significantly reduced when the plus-strand RNA wastargeted and miRNA inhibitors were not supplied at the time of RNAelectroporation of cells. In addition, miRNA targets in either a 5′ or3′ location relative to the capsid gene or in both the 5′ and 3′locations simultaneously were effective at reducing expression andreplication of the helper RNA in the absence of miRNA inhibitoraddition. As noted in previous experiments, VEE RP yields were 3 ordersof magnitude lower in cells that did not receive the miRNA inhibitorswhen compared to those that did (FIG. 12).

Experiments to determine contribution of individual miRNA targets onhelper RNA replication. To determine what effect each miRNA target wascontributing to the overall control of replication, six copies of eachindividual target miRNA sequence were cloned into the 3′ UTR of thecapsid helper plus-strand RNA. Capsid helper RNA and unmodified GPhelper RNA were combined with replicon RNA and electroporated into Verocells in the presence or absence of miRNA inhibitors specific for themiRNA targets present on the capsid helper. The electroporated cellswere seeded into media and incubated overnight. After incubation (˜18hours), samples were collected to examine capsid-specific proteinexpression (Western blot and IFA), RNA replication (Northern blot) andVEE RP yield. The results of capsid-specific Western and Northern blotanalysis are shown in FIG. 13 and FIG. 14, respectively. These resultsshow that cellular miRNA specific for let-7, miR-155, miR-17 and miR-19(RC1, RC4, RC5 and RC6, respectively) are capable of down-regulatinghelper expression and replication (in the absence of specific miRNAinhibitor) and that miRNA specific for lin-4 (RC2) and miR-101 (RC3) areless effective at controlling helper expression/replication in this Verocell packaging system (FIG. 13 and FIG. 14). The VEE RP yields producedusing the different miRNA targeted capsid helpers are shown in FIG. 15.The capsid helper containing all six miRNA targets (RC1-6) demonstratedthe largest difference in VEE RP yields in the presence and absence ofmiRNA inhibitor (3 orders of magnitude) indicating an additive effect ofthe miRNA targets on helper replication in this experiment. The timer ofmiRNA let-7 (RC1x6) target demonstrated the next largest effect on VEERP yield (˜2.5 orders of magnitude) with miR-155 (RC4x6) and miR-17(RC5x6) both demonstrating ˜1.5 orders of magnitude differences in VEERP yield. The helper with six copies of the miR-19 target (RC6x6)produced about one-half log less VEE RP yield while lin-4 (RC2x6) andmiR-101 (RC3x6) targets demonstrated little difference in VEE RP yield(FIG. 15). In combination, the Western, Northern and VEE RP yield dataindicate that these selected individual miRNA targets play a larger orsmaller role in the overall miRNA effect on helper replication in Verocell packaging, but all can contribute to the overall effect.

Experiments to determine copy number requirement of miRNA targets onhelper RNAs. The experiments described above suggested that each miRNAhad a different effect on miRNA-targeted helper replication (and VEE RPyield) and that some miRNA targets were more efficient at controllinghelper replication in Vero cells than others. Additional helperconstructs were produced using miRNA targets that imparted control ofRNA replication in Vero cells. Two miRNA targets, RC1 (let-7) and RC5(miR-155) were selected for these studies because they represent miRNAtargets with a strong and a medium effect on helper replication, in Verocells, respectively. The new capsid and GP helpers were constructed witheither a single copy or three copies of each individual miRNA target(RC1x1, RC1x3, RC5x1 and RC5x3) engineered into the 3′ UTR of theplus-strand RNA. These helpers were designed to determine what number ofmiRNA target copies, for these particular miRNAs, was required to impartRNA replication control in Vero cells.

Capsid and GP helper RNAs, with the same complement of miRNA targets,were combined with replicon RNA and electroporated into Vero cells inthe presence or absence of miRNA inhibitors specific for the miRNAtargets present on the helpers. In one set of electroporations the2′-O-methylated oligonucleotide miRNA inhibitor specific for let-7 wasreplaced with a phosphorodiamidate morpholino oligomer (PMO) specificfor let-7 miRNA to determine whether this type of molecule wouldfunction to block the action of the let-7 miRNA on the targeted helpers.The electroporated cells were seeded into media and incubated overnight.After incubation (˜18 hours), samples were collected to examinehelper-specific protein expression, RNA replication and VEE RP yield. Asfew as one copy of the let-7 or miR-155 miRNA target (RC1x1 or RC5x1,respectively) resulted in reduction of both protein expression (FIG. 16and FIG. 19) and RNA replication (FIG. 17 and FIG. 20) of both helpers.The reduction noted in protein expression and helper replication wasmirrored in the VEE RP yields produced from cells in the absence ofspecific inhibitor (FIG. 18 and FIG. 21). Helpers containing the let-7miRNA target (RC1) were more completely controlled (even with a singlecopy) than were helpers containing the miR-155 miRNA target(s). VEE RPyields in the absence of miRNA-specific inhibitor were reduced ˜3 ordersof magnitude using helpers with the RC1 target no matter what the copynumber was (FIG. 18). In contrast, there was a larger reduction in VEERP yields in the absence of miRNA-specific inhibitor using helpers witheither 3 or 6 copies of the miR-155 miRNA targets (5×3 and 5×6,respectively) than with a single miR-155 target (5×1) (FIG. 21). Thesedata indicate that more than one copy of the miR-155 target (RC5) wasrequired to have an effect similar to one copy of the let-7 target (RC1)for optimal RNA replication control in this Vero cell. In addition, thePMO-based let-7 miRNA inhibitor restored protein expression (FIG. 16)and RNA replication (FIG. 20) to levels similar to the rescue noted for2′-O-methylated oligonucleotide miRNA inhibitor. VEE RP yields producedin the presence of PMO-based inhibitor were nearly 2 orders of magnitudehigher than in the absence of inhibitor but not as great as those using2′-O-methylated oligonucleotide miRNA inhibitors (FIG. 18).

Experiments to demonstrate the functionality of miRNA targeted, cleavagedeficient, capsid helper RNAs. Experiments were conducted to demonstratethat the m152 capsid mutation 1) blocked production of VEE RP when themutation was introduced into the capsid gene in the context of a DNAhelper and 2) when introduced into an miRNA targeted capsid RNA helper,caused no or little reduction in VEE RP yield, when miRNA inhibitorswere included in the packaging cell system for producing VEE RP. Verocells were electroporated with replicon RNA and the following helpercombinations 1) pHCMV-Vsp, 2) pHCM-Vsp(m152), 3) dHcap6-mut1(W-stop)m152and dHgp6-mut1, 4) dHcap6-mut1(W-Stop)m152 RC4-6 and dHgp6-mut1, and 5)dHcap6-mut1(W-Stop)m152 RC4-6 and dHgp6-mut1 plus miRNA inhibitorsspecific for RC4-6. The VEE RP yields produced from each of thesecombinations are shown in the Table 2.

TABLE 2 VRP production with cleavage deficient helper constructs. RNAinhibitor combination Capsid helper GP helper added VRP/ml 1 pHCMV-VspNA no 7.2E+07 2 pHCM-Vsp(m152) NA no 0 3 dHcap6-mut1(W- dHgp6-mut1 no7.9E+08 stop)m152 4 dHcap6-mut1(W- dHgp6-mut1 no 1.4E+06 Stop)m152 RC4-65 dHcap6-mut1(W- dHgp6-mut1 yes 8.6E+08 Stop)m152 RC4-6

The data indicate that the H→G change at amino acid 152 of the capsidprotein blocks the production of VEE RP when the mutation is in thecontext of a DNA helper; in this context the capsid would rely on theautoprotease function to cleave itself from the co-translated VEEglycoproteins (compare RNA combination 1 and 2). In contrast, if them152 mutation is introduced into a capsid helper RNA (where theautoprotease activity is not required) VEE RP are produced (RNAcombination 3). Furthermore, introduction of ml 52 into miRNA targetedcapsid helper also has no negative affect on VEE RP yield as long asmiRNA inhibitor is added to the electroporation mix (compare RNAcombination 4 and 5).

Experiments to determine cell type specificity of miRNA activity onalphavirus RNA replication. All of the previous experiments wereconducted in Vero cells. To determine whether the miRNA targets we havetested in Vero cells have the same function in other cell types,replicon vectors that express the CAT reporter gene were constructedthat code for three or six copies of each individual miRNA target (e.g.,pERK/342/EMCV/CAT-RC1x6, pERK/342/EMCV/CAT-RC2x6,pERK/342/EMCV/CAT-RC7x3, etc.). VEE RP preparations were prepared byelectroporating Vero cells, in the presence of the relevant miRNAinhibitor, with each of the miRNA targeted CAT replicon RNAs combinedwith unmodified capsid and GP helper RNAs. To eliminate difficulty indetermining accurate Vero cell infectious unit (IU) titers of these VEERP preparations (due to the effect of the miRNAs present in Vero cellsreducing the effective IU titer) replicon genome equivalents (GE) weredetermined by quantitative reverse transcriptase PCR. Genome equivalenttiters were used to control the multiplicity of infection (MOI) for eachof the cell types analyzed. The MOI used was 3-5 in each of theseexperiments. A summary of the relative miRNA activity detected in eachcell type, based on the percent of CAT expressed from each of the miRNAtargeted replicons relative to an unmodified CAT replicon is shown inTable 3 and Table 4.

TABLE 3 MicroRNA Activity in Various Cell Types (RC 1-6). Cell typeHuman Hamster NHP Mouse Muscle Lung Musc. Hep. Kid. Ovary Kid. Bov.Neuron Cell name HEL-299 RD MO-59K MRC-5 SkM PHH BHK CHO Vero BT CNReplicon (miRNA) Relative % CAT Expression vs. CAT replicon control CAT(none) 100 100 100 100 100 100 100 100 100 100 100 CAT (RC1-6) 0 5 0 1nt nt 1 0 0 nt nt CAT (RC1x6) 0 20 0 0 1 0 0 0 0 0 6 CAT (RC2x6) 3 20 154 11 11 31 1 42 8 59 CAT (RC3x6) 26 41 50 49 38 0 86 36 37 50 70 CAT(RC4x6) 13 52 46 57 42 32 81 66 11 55 100 CAT (RC5x6) 7 0 6 35 14 0 13 01 18 2 CAT (RC6x6) 11 8 28 45 30 0 60 8 14 32 74

TABLE 4 MicroRNA Activity in Various Cell Types (RC 7-12) Cell typeHuman Mouse NHP Muscle Neuron Kidney Cell name RD SkM CN Vero RepliconRelative % CAT expression (miRNA) vs. CAT replicon control CAT (none)100 100 100 100 CAT (RC7x3) 49 10 80 55 CAT (RC8x3) 2 6 39 4 CAT (RC9x3)3 6 1 32 CAT (RC10x3) 100 31 46 54 CAT (RC11x3) 15 100 100 100 CAT(RC12x3) 11 54 17 100

VEE RP generated with a CAT replicon carrying all six of the miRNAtargets (CAT 1-6) expressed very little CAT protein in any cell typetested. Similarly, minimal CAT protein could be detected in cellsinfected with VEE RP containing a replicon RNA carrying the let-7 miRNAtarget (CAT RC1x6). These results show that the let-7 miRNA is presentin all of the cell types tested. CAT protein levels detected in cellsinfected with the other individual miRNA targeted CAT repliconsindicated a range of protein expression. These data show that eachdifferent cell type had a different complement of active miRNAsavailable to control replication of replicon RNA.

Experiments to determine in vivo miRNA activity on alphavirus RNAreplication. The data described above indicate that the miRNA-targetedhelper and replicon RNAs can be controlled by the action of cellularmiRNAs in a wide array of cells in culture (in vitro). To determinewhether the same miRNA control demonstrated in vitro could bedemonstrated in vivo, a number of VEE RP preparations were produced andtested in BALB/c mice. Combinations of both miRNA-targeted helpers weremixed with unmodified or miRNA-targeted replicon vectors expressing theinfluenza HA gene (A/Wisconsin). A summary of the helper and repliconRNA combinations is shown in Table 5.

TABLE 5 Summary of Helper and Replicon RNA Combinations. GroupIdentifier Replicon RNA Capsid Helper GP Helper Wild typepERK/342/EV71/A(Wis)/HA dHcap6-mut1 dHgp6-mut1 (“WT”) (W-stop) miCappERK/342/EV71/A(Wis)/HA dHcap6-mut1 dHgp6-mut1 (W-stop)RC1-6 miGPpERK/342/EV71/A(Wis)/HA dHcap6-mut1 dHgp6- (W-stop) mut1-RC1-6 miCap +pERK/342/EV71/A(Wis)/HA dHcap6-mut1 dHgp6- miGP (W-stop)RC1-6 mut1-RC1-6miRep + pERK/342/EV71/A(Wis)/HA- dHcap6-mut1 dHgp6- miCap + RC1-6(W-stop)RC1-6 mut1-RC1-6 miGP

Groups of 16 mice were immunized with equivalent doses of each of theVEE RP. Seven days after the priming dose, half (8) of the mice weresacrificed, the splenocytes were collected and HA-specific gammainterferon ELISPOT analysis was conducted. The results of the 7 daypost-prime HA-specific ELISPOT analysis are shown in FIG. 22. There weresignificantly less spot forming cells (SFCs) in the group immunized withVEE RP generated with miRNA-targeted replicon and helper RNAs(“miRep+miCap+miGP”) than in the groups immunized with VEE RP generatedwith unmodified replicon and helper RNAs (“WT”) or with VEE RP generatedwith unmodified replicon RNA and miRNA-targeted capsid and GP helpers(“miCap+miGP”). Three weeks after the priming dose, the remaining micein each group were boosted with their respective HA VEE RP. Seven daysafter the boost the remaining animals were sacrificed and thesplenocytes were collected for HA-specific gamma interferon ELISPOTanalysis. The post boost HA-specific ELISPOT analysis are shown in FIG.23. After the boost, the SFCs detected in the group immunized with VEERP generated using miRNA-targeted replicon RNA were significantly lowerthan those detected in all of the other VEE RP immunized groups.Furthermore, HA-specific ELISA analysis of both post prime (FIG. 24) andpost boost (FIG. 25) serum samples demonstrated that animals immunizedwith VEE RP generated with an miRNA-targeted replicon were significantlylower than all other VEE RP immunized groups. These data show that themiRNA-targeted replicon VEE RP were significantly inhibited frominducing an HA specific cellular or humoral immune response.

A second mouse study was conducted using VEE RP generated withmiRNA-targeted helper RNAs. A different complement of miRNA targets wasengineered into the 3′ UTR of RNA helpers used in this study. The capsidand gp helper RNAs contained the following miRNA targets: RC1, RC4, RC5,RC9, RC2, RC3, RC10, RC7 and RC12(5′-gcatgcaactatacaacctactacctcacccctatcacgattagcattaaactacctgcactgtaagcactttgactcaccgacagcgttgaatgttacacagtcgaaggtctcagggacttcagttatcacagtactgtaggcattcaccgcgtgccttagagctacagtgcttcatctcacagctggttgaaggggaccaaagtttaaac-3′(SEQ ID NO:39)). A replicon vector expressing the herpes simplex virusII gD (HSV gD) gene was packaged into VEE RP with 4 differentcomplements of helper RNAs as shown in Table 6.

TABLE 6 Replicon and helper RNA combinations used to generate HSV VRP.Group indentifier Replicon RNA Capsid helper GP helper Wild type pAVP1HSV dHcap6-mut1(W-stop) dHgp6-mut1 152 pAVP1 HSV dHcap6-mut1(W-stop)152dHgp6-mut1 mi pAVP1 HSV dHcap6-mut1(W-stop)mi dHgp6-mut1 mi mi152 pAVP1HSV dHcap6-mut1(W-stop)mi152 dHgp6-mut1 mi

Groups of 6 mice were immunized with 5×10⁶ IU of each VEE RP at 0 and 3weeks. No difference was noted in the HSV gD-specific humoral orcellular responses detected in animals immunized with HSV VEE RPpackaged with the different helper RNA combinations. The anti-VEE RPimmune response, also referred to as an “anti-vector response”, elicitedin the vaccinated animals was also determined. The results of VEE RPneutralization assays carried out with serum collected from vaccinatedanimals is shown in FIG. 26. Animals vaccinated with VEE RP generatedwith miRNA-targeted helper RNAs demonstrated lower anti-VEE RPneutralization titers than animals vaccinated with VEE RP generated withhelper RNAs that did not code for miRNA targets. These data indicatethat it is possible to reduce anti-vector immune responses by packagingVEE RP with miRNA-targeted helper RNAs.

Taken together, the two mouse studies presented above indicate thatmiRNA target-specific inhibition of both replicon and helper RNAreplication is occurring in vivo. These results are in completeagreement with the miRNA target-specific inhibition that wasdemonstrated in vitro in the absence of miRNA inhibitors. The in vitroand in vivo data presented above show that engineering miRNA targetsinto alphavirus helper RNAs controls replication/expression of theseRNAs in cells where helper function is not required or wanted.

Efficacy of VRP Made with miRNA-Targeted Helpers in Non-Human Primates

Replicon vectors were constructed using the L1R gene from vaccinia virus(VACV) which is a homolog to a similar gene in smallpox. Two differentalphavirus vector systems were used: “pVEK” which is derived from TC83,and “pERK” which is derived from attenuated VEE strain V3014. The L1Rgene was modified to include a TPA signal sequence which enhances theimmunogenicity of the construct. L1R-expressing VEE RP were generatedusing miRNA targeted helpers (described hereinabove asdHcap6-mut1(W-stop)mi152 and dHgp6-mut1mi).

Groups of five Cynomolgus macaques were vaccinated by the intramuscularroute on week 0 and 28 as follows: Group 1, 5×10⁷ IU of pVEK L1R-VRP;Group 2, a combination of 2.5×10⁷ IU of pVEK L1R-VRP and 2.5×10⁷ IU ofpERK L1R VRP; and group 3, 5×10⁷ IU of pVEK L1R-VRP. Serum and PBMCsamples were collected from animals on day 0, 21 (prime), 35 (1 wk PB)and 56 (4 wk PB). The results are summarized in Table 7.

TABLE 7 Summary of results for vaccinated Cynomolgus macaques. Log ELISAGMT PRNT₅₀ Mean L1R Specific SFC^(c) Group VRP Vaccine prime 1 wk PB^(a)4 wk PB GMT^(b) prime 1 wk PB^(a) 4 wk PB 1 pVEK L1R 3.4 5.6 5.0 183.812 109 155 2 pVEK L1R + 3.8 5.1 4.2 211.1 9 34 18 pERK L1R 3 pERK L1R3.5 5.1 4.6 139.3 15 24 59 ^(a)post-boost ^(b)VacV neutralizing antibodygeometric mean titer ^(c)per 10⁶ PBMC (background subtracted)

The immune responses detected in these macaques were consistent with theresponses previously detected in animals receiving a mixture ofL1R-expressing VEE RP and 3 other VACV gene-expressing VEE RP in achallenge study in which vaccinated macaques were significantlyprotected from lethal disease. These data indicate that VRP generatedwith miRNA targeted helpers are immunogenic in non-human primates.

What is claimed is:
 1. A helper nucleic acid comprising: (a) a 5′alphavirus replication recognition sequence; (b) a nucleic acid sequenceencoding an alphavirus structural protein; (c) a 3′ alphavirusreplication recognition sequence; and (d) at least one microRNA targetsequence of a cellular microRNA wherein the helper nucleic acid is not alive, attenuated virus.
 2. The helper nucleic acid of claim 1, whereinthe microRNA target sequence has at least 70% complementarity to thecellular microRNA.
 3. The helper nucleic acid of claim 1, wherein atleast one microRNA target sequence is located in a 3′ UTR or 5′ UTR ofthe nucleic acid sequence encoding the alphavirus structural protein. 4.The helper nucleic acid of claim 3, wherein at least one microRNA targetsequence is located in a 3′ UTR of the nucleic acid sequence encodingthe alphavirus structural protein and at least one microRNA targetsequence is located in a 5′ UTR of the nucleic acid sequence encodingthe alphavirus structural protein.
 5. The helper nucleic acid of claim1, wherein at least one microRNA target sequence is located in thetranslated region of the nucleic acid sequence encoding the alphavirusstructural protein.
 6. The helper nucleic acid of claim 1, wherein thealphavirus structural protein is a Venezuelan equine encephalitis (VEE)virus structural protein.
 7. The helper nucleic acid of claim 1, whereinthe alphavirus structural protein is selected from the group consistingof South African Arbovirus No. 86, Sindbis virus, Semliki Forest Virus,and Ross River Virus structural proteins.
 8. The helper nucleic acid ofclaim 1, wherein the alphavirus structural protein is an alphaviruscapsid protein with reduced autoprotease activity.
 9. The helper nucleicacid of claim 8, wherein the alphavirus capsid protein is a VEE capsidprotein.
 10. The helper nucleic acid of claim 9, wherein the VEE capsidprotein comprises one or more amino acid substitutions at residues 152,174, or
 226. 11. The helper nucleic acid of claim 1, wherein the helpernucleic acid comprises at least two microRNA target sequences.
 12. Acomposition comprising: (a) a first helper nucleic acid according toclaim 1, and (b) an alphavirus replicon.
 13. The composition of claim12, further comprising a second helper nucleic acid comprising at leastone microRNA target sequence of a cellular microRNA and a nucleic acidsequence encoding an alphavirus structural protein, wherein the secondhelper nucleic acid encodes at least one or more alphavirus structuralproteins not encoded by the first helper nucleic acid.
 14. A cellcomprising one or more helper nucleic acids of claim 1, wherein the cellis stably transformed with at least one helper nucleic acid.
 15. A cellcomprising (a) a first helper nucleic acid according to claim 1, and (b)a replicon.
 16. The cell of claim 15, further comprising a second helpernucleic acid comprising at least one microRNA target sequence of acellular microRNA and a nucleic acid sequence encoding an alphavirusstructural protein, wherein the second helper nucleic acid encodes atleast one or more alphavirus structural proteins not encoded by thefirst helper nucleic acid.
 17. The cell of claim 15, further comprisingan inhibitor of at least one of the cellular microRNAs.
 18. The cell ofclaim 17, further comprising a nucleic acid encoding the inhibitor ofthe cellular microRNA.
 19. A population of alphavirus-like repliconparticles (ARPs) comprising (i) a first subset of particles comprising areplicon and (ii) a second subset of particles comprising the helpernucleic acid of claim 1 or a fragment thereof, wherein the fragmentcomprises at least one microRNA target sequence, and a replicon.
 20. Thepopulation of claim 19, wherein the ARPs are Venezuelan equineencephalitis (VEE) virus ARPs.
 21. The population of claim 19, whereinthe ARPs are selected from the group consisting of South AfricanArbovirus No. 86, Sindbis virus, Semliki Forest Virus, and Ross RiverVirus ARPs.
 22. A method of making alphavirus-like replicon particles(ARPs) comprising: (a) transfecting a cell with (i) a replicon, and (ii)one or more helper nucleic acids according to claim 1, wherein thealphavirus structural proteins necessary to make the ARPs are encoded byone or more of the cell, the replicon or the one or more helper nucleicacids; and (b) culturing the cell under conditions that allow forproduction of assembled ARPs comprising the replicon; and (c) collectingthe ARPs.
 23. The method of claim 22, wherein the cell is transfectedwith a first helper nucleic acid and a second helper nucleic acid,wherein the first and second helper nucleic acids encode the alphavirusstructural proteins necessary to make ARPs and wherein the second helpernucleic acid encodes at least one or more alphavirus structural proteinsnot encoded by the first helper nucleic acid.
 24. The method of claim22, wherein the cell is cultured in the presence of an inhibitor to atleast one of the cellular microRNAs.
 25. The method of claim 22, whereinthe ARPs are Venezuelan equine encephalitis (VEE) virus ARPs.
 26. Themethod of claim 22, wherein the ARP is selected from the groupconsisting of South African Arbovirus No. 86, Sindbis virus, SemlikiForest Virus, and Ross River Virus ARPs.
 27. A method of inducing animmune response in a subject comprising administering to the subject thepopulation of alphavirus-like replicon particles of claim
 19. 28. Themethod of claim 22, wherein the cell does not contain the cellularmicroRNA.
 29. A helper nucleic acid comprising: (a) a 5′ alphavirusreplication recognition sequence; (b) a nucleic acid sequence encodingan alphavirus structural protein; (c) a 3′ alphavirus replicationrecognition sequence; and (d) at least one microRNA target sequence of acellular microRNA wherein the helper nucleic acid is propagationdefective.
 30. A composition comprising the helper nucleic acid of claim29 and an alphavirus replicon.
 31. A cell comprising the helper nucleicacid of claim 29 and an alphavirus replicon.